Opioid receptor genes

ABSTRACT

Genes encoding opioid receptors can be retrieved from vertebrate libraries using the murine probe disclosed herein under low-stringency conditions. The DNA sequence shown in FIG.  5  or its complement can be used to obtain the human delta, kappa and mu genes as well as the murine mu gene. The probe provided encodes the murine delta opioid receptor.

This application is filed under 35 U.S.C. § 371 as a national phase ofPCT/US93/07665 filed Aug. 13, 1993 which is a continuation-in-part ofU.S. Ser. No. 07/929,200 filed Aug. 13, 1992, now abandoned.

This invention was made with Government support under Grant No. DA05010awarded by the Alcohol, Drug Abuse and Mental Health Administration. TheGovernment has certain rights in the invention.

TECHNICAL FIELD

The invention relates to substances involved in vertebrate nervoussystems, and in particular to the opioid receptors and activitiesmediated thereby. Accordingly, the invention concerns recombinantmaterials useful for the production of opioid receptors, the receptor asa diagnostic tool, therapeutic and diagnostic compositions relevant tothe receptor, and methods of using the receptor to screen for drugs thatmodulate the activity of the receptor.

BACKGROUND ART

The term “opioid” generically refers to all drugs, natural andsynthetic, that have morphine-like actions. Formerly, the term “opiate”was used to designate drugs derived from opium, e.g., morphine, codeine,and many semi-synthetic congeners of morphine. After the isolation ofpeptide compounds with morphine-like actions, the term opioid wasintroduced to refer generically to all drugs with morphine-like actions.Included among opioids are various peptides that exhibit morphine-likeactivity, such as endorphins, enkephalins and dynorphins. However, somesources have continued to use the term “opiate” in a generic sense, andin such contexts, opiate and opioid are interchangeable. Additionally,the term opioid has been used to refer to antagonists of morphine-likedrugs as well as to characterize receptors or binding sites that combinewith such agents.

Opioids are generally employed as analgesics, but they may have manyother pharmacological effects as well. Morphine and related opioidsproduce their major effects on the central nervous and digestivesystems. The effects are diverse, including analgesia, drowsiness, moodchanges, respiratory depression, dizziness, mental clouding, dysphoria,pruritus, increased pressure in the biliary tract, decreasedgastrointestinal motility, nausea, vomiting, and alterations of theendocrine and autonomic nervous systems.

A significant feature of the analgesia produced by opioids is that itoccurs without loss of consciousness. When therapeutic doses of morphineare given to patients with pain, they report that the pain is lessintense, less discomforting, or entirely gone. In addition toexperiencing relief of distress, some patients experience euphoria.However, when morphine in a selected pain-relieving dose is given to apain-free individual, the experience is not always pleasant; nausea iscommon, and vomiting may also occur. Drowsiness, inability toconcentrate, difficulty in mentation, apathy, lessened physicalactivity, reduced visual acuity, and lethargy may ensue.

The development of tolerance and physical dependence with repeated useis a characteristic feature of all opioid drugs, and the possibility ofdeveloping psychological dependence on the effect of these drugs is amajor limitation for their clinical use. There is evidence thatphosphorylation may be associated with tolerance in selected cellpopulations (Louie, A. et al. Biochem Biophys Res Comm (1988)152:1369-75).

Acute opioid poisoning may result from clinical overdosage, accidentaloverdosage, or attempted suicide. In a clinical setting, the triad ofcoma, pinpoint pupils, and depressed respiration suggest opioidpoisoning. Mixed poisonings including agents such as barbiturates oralcohol may also contribute to the clinical picture of acute opioidpoisoning. In any scenario of opioid poisoning, treatment must beadministered promptly.

The opioids interact with what appear to be several closely relatedreceptors. Various inferences have been drawn from data that haveattempted to correlate pharmacologic effects with the interactions ofopioids with a particular constellation of opioid receptors (Goodman andGilman's, THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th ed, pp. 493-95(MacMillan 1985)). For example, analgesia has been associated with muand kappa receptors. Delta receptors are believed to be involved inalterations of affective behavior, based primarily on the localizationof these receptors in limbic regions of the brain. Additionally,activation, e.g., ligand binding with stimulation of furtherreceptor-mediated responses, of delta opioid receptors is believed toinhibit the release of other neurotransmitters. The pathways containingrelatively high populations of delta opioid receptor are similar to thepathways implicated to be involved in Huntington's disease. Accordingly,it is postulated that Huntington's disease may correlate with someeffect on delta opioid receptors.

Two distinct classes of opioid molecules can bind opioid receptors: theopioid peptides (e.g., the enkephalins, dynorphins, and endorphins) andthe alkaloid opiates (e.g., morphine, etorphine, diprenorphine andnaloxone). Subsequent to the initial demonstration of opiate bindingsites (Pert, C. B. and Snyder, S. H., Science (1973) 179:1011-1014), thedifferential pharmacological and physiological effects of both opioidpeptide analogues and alkaloid opiates served to delineate multipleopioid receptors. Accordingly, three anatomically and pharmacologicallydistinct opioid receptor types have been described: delta, kappa and mu.Furthermore, each type is believed to have sub-types (Wollemann, M., J.Neurochem. (1990) 54:1095-1101; Lord, J. A., et al., Nature (1977)267:495-499).

All three of these opicid receptor types appear to share the samefunctional mechanisms at a cellular level. For example, the opioidreceptors cause inhibition of adenylate cyclase, and inhibition ofneurotransmitter release via both potassium channel activation andinhibition of Ca²⁺ channels (Evans, C. J., In: Biological Basis ofSubstance Abuse, S. G. Korenman & J. D. Barchas, eds., Oxford UniversityPress (in press); North, A. R., et al., Proc Natl Acad Sci USA (1990)87:7025-29; Gross, R. A., et al., Proc Natl Acad Sci USA (1990)87:7025-29; Sharma, S. K., et al., Proc Natl Acad Sci USA (1975)72:3092-96). Although the functional mechanisms are the same, thebehavioral manifestations of receptor-selective drugs differ greatly(Gilbert, P. E. & Martin, W. R., J Pharmacol Exp Ther (1976) 198:66-82).Such differences may be attributable in part to the anatomical locationof the different receptors.

Delta receptors have a more discrete distribution within the mammalianCNS than either mu or kappa receptors, with high concentrations in theamygdaloid complex, striatum, substantia nigra, olfactory bulb,olfactory tubercles, hippocampal formation, and the cerebral cortex(Mansour, A., et al., Trends in Neurosci (1988) 11:308-14). The ratcerebellum is remarkably devoid of opioid receptors including deltaopioid receptors.

Several opioid molecules are known to selectively or preferentially binddelta receptors. Of the vertebrate endogenous opioids, the enkephalins,particularly met-enkephalin (SEQ ID NO:1) and leu-enkephalin (SEQ IDNO:2), appear to possess the highest affinity for delta receptors,although the enkephalins also have high affinity for mu receptors.Additionally, the deltorphans, peptides isolated from frog skin,comprise a family of opioid peptides that have high affinity andselectivity for delta receptors (Erspamer, V., et al., Proc Natl AcadSci USA (1989) 86:5188-92).

A number of synthetic enkephalln analogues are also deltareceptor-selective including (D-Ser²) leucine enkephalin Thr (DSLET)(SEQ ID NO:3) (Garcel, G. et al. (1980) FEBS. Lett. 118:245-247) and(D-Pen², D-Pen⁵) enkephalin (DPDPE) (SEQ ID NO:4) (Akiyama, K. et al.,Proc. Natl. Acad. Sci. USA (1985) 82:2543-2547).

Recently a number of other selective delta receptor ligands have beensynthesized, and their bioactivities and binding characteristics suggestthe existence of more than one delta receptor subtype (Takemori, A. E.,et al., Ann. Rev. Pharm. Toxicol., (1992) 32:239-69; Negri, L., et al.,Eur J Pharmacol (1991) 196:355-335; Sofuoglu, M., et al.,Pharmacolocrist (1990) 32:151).

Although the synthetic pentapeptide 2dAla, 5dLeu enkephalin (DADLE) (SEQID NO:5) was considered to be delta-selective, it also binds equallywell to mu receptors. The synthetic peptideD-Ala²-N-Me-Phe⁴-Gly-ol⁵-enkephalin (DAGO) (SEQ ID NO:6) has been foundto be a selective ligand for mu-receptors.

The existence of multiple delta opioid receptors has been implied notonly from the pharmacological studies addressed above, but also frommolecular weight estimates obtained by use of irreversible affinityligands. Molecular weights for the delta opioid receptor that range from30 kDa to 60 kda (Evans, C. J., supra; Evans, C. J. et al., Science258:1952-1955 (1992), which document corresponds to the dislcosure ofthe priority document of the present application; Bochet, P. et al., MolPharmacol (1988) 34:436-43). The various receptor sizes may representalternative splice products, although this has not been established.

Many studies of the delta opioid receptor have been performed with theneuroblastoma/glioma cell line NG108-15, which was generated by fusionof the rat glial cell line (C6BU-1) and the mouse neuroblastoma cellline (N18-TG2) (Klee, W. A. and Nirenberg, M. A., Proc. Natl. Acad. Sci.USA (1974) 71:3474-3477). The rat glial cell line expresses essentiallyno delta opioid receptors, whereas the mouse neuroblastoma cell lineexpresses low amounts of the receptor. Thus, it has been suggested thatthe delta receptor in the NG108-15 cells is of mouse chromosomal origin(Law, Mol Pharm (1982) 21:438-91). Each NG108-15 cell is estimated toexpress approximately 300,000 delta-receptors. Only delta-type opioidreceptors are expressed, although it is not known whether theserepresent more than a single subtype. Thus, the NG108-15 cell line hasserved to provide considerable insight into the binding characterizationof opioid receptors, particularly delta opioid receptors. However, theNG108-15 cell line is a cancer-hybrid and may not be completelyrepresentative of the delta receptor in endogenous neurons due to theunique cellular environment in the hybrid cells.

An extensive literature has argued that the opioid receptors are coupledto G-proteins (see, e.g., Schofield, P. R., et al., EMBO J., 8:489-95(1989)), and are thus members of the family of G-protein coupledreceptors. G-proteins are guanine nucleotide binding proteins thatcouple the extracellular signals received by cell surface receptors tovarious intracellular second messenger systems. Identified members ofthe G-protein-coupled family share a number of structural features, themost highly conserved being seven apparent membrane-spanning regions,which are highly homologous among the members of this family (Strosberg,A. D., Eur. J. Biochem. 196:1-10 (1991)). Evidence that the opioidreceptors are members of this family includes the stimulation of GTPaseactivity by opioids, the observation that GTP analogues dramaticallyeffect opioid and opiate agonist binding, and the observation thatpertussis toxin (which by ADP ribosylation selectively inactivates boththe Gi and Go subfamilies of G-proteins) blocks opioid receptor couplingto adenylate cyclase and to K⁺ and Ca²⁺ channels (Evans, C. J., supra).

The members of the G-protein-coupled receptor family exhibit a range ofcharacteristics. Many of the G-protein-coupled receptors, e.g., thesomatostatin receptor and the angiotensin receptor, have a single exonthat encodes the entire protein coding region (Strosberg supra; Langord,K., et al., Biochem. Biophys. Res. Comm. 138:1025-1032 (1992)). However,other receptors, such as substance P receptor and the dopamine D2receptor, contain the protein coding region. The D2 receptor isparticularly interesting in that alternate splicing of the gene givesrise to different transcribed products (i.e., receptors) (Evans, C. J.,supra; Strosberg, supra). Interestingly, somatostatin ligands arereported to bind to opioid receptors (Terenius, L., Eur. J. Pharmacol.38:211 (1976); Mulder, A. H., et al., Eur. J. Pharmacol. 205:1-6 (1991))and, furthermore, to have similar molecular mechanisms (Tsunoo, A., etal., Proc. Natl. Acad. Sci. USA 83:9832-9836 (1986)).

In previous efforts to describe and purify opioid receptors, two cloneshave been described that were hypothesized either to encode a portion ofor entire opioid receptors. The first clone, which encodes the opiatebinding protein OBCAM (Schofield et al., supra), was obtained byutilizing a probe designed from an amino acid sequence of a proteinpurified on a morphine affinity column. OBCAM lacks any membranespanning domains but does have a C-terminal domain that ischaracteristic of attachment of the protein to the membrane by aphosphatidylinositol (PI) linkage. This feature, which is shared bymembers of the immunoglobulin superfamily, is not common to the familyof receptors coupled to G-proteins. Thus, it has been proposed thatOBCAM is part of a receptor complex along with other components that arecoupled to G-proteins (Schofield et al., supra). At present, however,there is no direct evidence for such a complex.

A second proposed opioid receptor clone was obtained in an effort toclone a receptor that binds kappa opioid receptor ligands (Xie, G. X.,Proc Natl Acad Sci USA 89:4124-4128 (1992)). A DNA molecule encoding aG-coupled receptor from a placental cDNA library was isolated. Thisreceptor has an extremely high homology with the neurokinin B receptor(84% identical throughout the proposed protein sequence). When thisclone was expressed in COS cells, it displayed opioid peptidedisplaceable binding of ³H-bremazocine (an opiate ligand with highaffinity for kappa receptors). However, the low affinity of thisreceptor for ³H-bremazocine, and the lack of appropriate selectivitysince this receptor (binding both mu and delta ligands) made it doubtfulthat this cloned molecule is actually an opioid receptor.

Furthermore, characterization of opioid receptor proteins has provendifficult because of their instability once solubilized from themembrane; purified delta opioid receptors have not been isolated. Theprevious estimates of opioid receptor molecular weights ranging from 30kDa to 60 Kda further reflect the difficulty in isolating andcharacterizing these proteins.

Recently, DNA encoding the murine kappa and delta opioid receptors frommouse brain was reported by Yasuda, K. et al. Proc Natl Acad Sci USA(1993) 90:6736-6740. The sequence of the clones indicated the presenceof the expected seven transmembrane regions. In addition, Chen, Y. etal. in a soon-to-be-published manuscript in Molecular Pharmacology(1993) report the “molecular cloning and functional expression of a muopioid receptor from rat brain”. In fact, the rat mu receptor was clonedusing the present inventors' DOR-1 clone, which lends enabling supportto the present invention disclosed below. Finally, the mouse deltaopioid receptor was disclosed as having been cloned (Kieffer, B. J. etal., Proc. Natl. Acad. Sci. USA 89:12048-12052 (1992 Dec.) after thefiling date of the priority document of the present application.However, the sequence reported therein differs from the sequencereported by the present inventors for the mouse delta receptor (Evans etal., 1992, supra; this disclosure).

DISCLOSURE OF THE INVENTION

The present invention provides recombinant nucleic acid molecules whichencode the murine delta opioid receptor, as well as recombinant nucleicacid molecules which can be retrieved using low-stringency hybridizationto this disclosed DNA. Thus, the invention provides genes encoding thedelta, kappa and mu receptors of any species containing genes encodingsuch receptors sufficiently homologous to hybridize under low-stringencyconditions described herein.

Thus, in one aspect, the invention is directed to recombinant nucleicacid molecules and methods for the production of an opioid receptorwherein the opioid receptor is encoded by a gene which hybridizes underlow-stringency to the nucleotide sequence of FIG. 5 (SEQ ID NO:7) or toits complement. By “low-stringency” is meant 50% formamide/6×SSC,overnight at 37° C. for the hybridization, followed by washes at 2×SSC0.1% SDS at room temperature.

Also provided are expression systemsd comprising the nucleic acidmolecules described above. The receptor can be recombinantly producedusing these expression systems and host cells modified to contain them.

Especially useful are vertebrate cells which express the opioid receptorgene so that the opioid receptor protein is displayed at the surface ofthe cells. These cells offer means to screen native and syntheticcandidate agonists and antagonists for the opioid receptors.

In still other aspects, the invention is directed to methods to screencandidate agonists and/or antagonists acting at opioid receptors usingthe recombinant transformed cells of the invention. Such assays include(1) binding assays using competition with ligands known to bind opioidreceptors, (2) agonist assays which analyze activation of the secondarypathways associated with opioid receptor activation in the transformedcells, and (3) assays which evaluate the effect on binding of thecandidate to the receptor by the presence or absence of sodium ion andGTP. Antagonist assays include the combination of the ability of thecandidate to bind the receptor while failing to effect furtheractivation, and, more importantly, competition with a known agonist.

Still another aspect of the invention is provision of antibodycompositions which are immunoreactive with the opioid receptor proteins.Such antibodies are useful, for example, in purification of thereceptors after solubilization or after recombinant production thereof.

In still other aspects, the invention is directed to probes useful forthe identification of DNA which encodes related opioid receptors invarious species or different types and subtypes of opioid receptors.

Accordingly, an object of the present invention is to provide anisolated and purified form of a DNA sequence encoding an opioidreceptor, which is useful as a probe as well as in the production of thereceptor.

Another object is to provide a recombinantly produced DNA sequenceencoding an opioid receptor.

Another object is to produce an antisense sequences corresponding toknown sense sequences encoding the opioid receptors.

Another object of the invention is to provide a DNA construct comprisedof a control sequence operatively linked to a DNA sequence which encodesan opioid receptor and to provide recombinant host cells modified tocontain the DNA construct.

Another object is to isolate, clone and characterize, from variousvertebrate species, DNA sequences encoding the various relatedreceptors, by hybridization of the DNA derived from such species with anative DNA sequence encoding the opioid receptor of the invention.

An advantage of the present invention is that opioid receptor-encodingDNA sequences can be expressed at the surface of host cells which canconveniently be used to screen drugs for their ability to interact withand/or bind to the receptors.

These and other objects, advantages and features of the presentinvention will become apparent to those persons skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a comparison of binding of ³H-diprenorphine (saturationcurves) between NG108-15 cells and COS cells three days followingtransfection (by electroporation) of each with DOR-1 in the CDM8 vector.Specific opioid binding was undetectable in nontransfected COS cells orCOS cells transfected with plasmid alone.

FIG. 2 depicts displacement curves of 5 nM ³H-diprenorphine from COScell membranes of cells transfected with DOR-1. ³H-diprenorphine wasdisplaced by diprenorphine, etorphine, morphine and levorphanol, but notby dextrorphan (the non-opiate active optical isomer of levorphanol).

FIG. 3 depicts displacement curves of 5 nm ³H-diprenorphine from COScell membranes of cells transfected with DOR-1. ³H-diprenorphine wasdisplaced by DPDPE and DSLET, which are delta-selective agonists, byDADLE, a high affinity ligand for mu and delta receptors, and bydynorphin 1-17, a kappa-preferring ligand. ³H-diprenorphine was notdisplaced by DAGO, a mu-selective ligand.

FIG. 4 depicts the results of a Northern analysis of mRNA from NG108-15cells and cells from various rat brain regions.

FIG. 5 shows the nucleotide sequence (SEQ ID NO:7) and the deduced aminoacid sequence (SEQ ID NO:8) of the DOR-1 clone.

FIG. 6 depicts the deduced amino acid sequence of DOR-1(SEQ ID NO:8),compared with the rat somatostatin receptor (SEQ ID NO:9). Consensusglycosylation sites predicted to fall in extracellular domains areindicated by an asterisk. Potential protein kinase C sites are listed inExample 5. The seven predicted membrane spanning regions (underlined)are predicted based on the hydrophobicity profile and publishedpredictions (Macvector software program (IBI); T. Hopp, and K. Woods,Proc Natl Acad Sci USA 78:3842-3828 (1981)). For sequencing, the cDNAinsert was subcloned into pBluescript and both strands were sequencedfrom single-stranded DNA using Sequenase and Taq cycle sequencing. Forambiguities due to compressions 7-deaza-dGTP replaced dGTP in thesequencing reactions and the products were resolved on formamide gels.

FIG. 7 depicts a Southern blot of radiolabeled DOR-1 cDNA probehybridized at high stringency to NG108-15, mouse, rat and human DNA cutwith BamHI.

FIG. 8a (SEQ ID NO:10 and SEQ ID NO:11) shows a partial nucleotidesequence of the human delta opioid receptor genomic clone H3 (alsodesignated human DORa or hDORa).

FIG. 8b (SEQ ID NO:12) shows a partial nucleotide sequence of the humankappa opioid receptor genomic clone H14 (also designated human KORa orhKORa).

FIG. 8c (SEQ ID NO:13) shows a partial nucleotide sequence of the humanmu opioid receptor genomic clone H20 (also designated human MORa orhMORa).

FIG. 8d (SEQ ID NO:14) shows the nucleotide sequence of the CACACArepeat near the H20 DNA.

FIG. 9 (SEQ ID NO:15) shows the nucleotide sequence of the murinemu-receptor clone DOR-2 also named mMOR-1 or mMOR-1α.

FIG. 10 (SEQ ID NO:8 and SEQ ID NO:16 and SEQ ID NO:17) shows thehomology of various receptor amino acid sequences.

MODES OF CARRYING OUT THE INVENTION

The invention provides DNA encoding mammalian opioid receptor proteinand additional recombinant nucleic acids, expression vectors and methodsuseful for the production of these proteins. In addition, eucaryoticcells, such as COS cells, transformed with the recombinant molecules ofthe invention so as to express opioid receptor proteins at their surfaceare useful in screening assays to identify candidate opioid agonists andantagonists. In addition, antibodies may be raised to the recombinantlyproduced opioid receptor proteins. These antibodies are useful inimmunoassays for said protein and in affinity purification thereof.

Recombinant Opioid Receptor

Illustrated hereinbelow is the obtention of a cDNA encoding a murinedelta opioid receptor. The complete DNA sequence of the cDNA, and theamino acid sequence encoded thereby, are set forth herein in FIG. 5. Theavailability of this cDNA permits the retrieval of the correspondingopioid receptor-encoding DNA from other vertebrate species. Accordingly,the present invention places within the possession of the art,recombinant molecules and methods for the production of cells expressingopioid receptors of various types and of various vertebrate species.Thus, the cDNA of FIG. 5, or a portion thereof, may be used as a probeto identify that portion of vertebrate genomic DNA or cDNA which encodesan opioid receptor protein. Illustrative methods used to prepare agenomic library and identify the opioid receptor-encoding genes aredescribed for convenience hereinbelow.

The DOR-1 clone described in FIG. 5 is a cDNA clone corresponding to themurine delta opioid receptor. The present inventors found, and describeherien, that screening of a human genomic library under conditions oflow stringency results in the recovery of DNA encoding all three typesof human opioid receptors. Similarly, a murine genomic clone wasobtained. In addition, a cDNA clone was obtained from a mouse brainlibrary encoding the murine mu opioid receptor. Thus, either cDNAlibraries from appropriate sources, such as brain, or genomic libraries,are fruitful sources or substrates for obtaining the DNA of the presentinvention and the corresponding recombinant materials. The invention isthus directed to DNA encoding an opioid receptor of a vertebrate,wherein the opioid receptor is encoded by a nucleotide sequence whichhybridizes under conditions of low stringency to the nucleotide sequenceshown in FIG. 5 or to its complement.

In the alternative, the DNA of FIG. 5 or a portion thereof may be usedto identify specific tissues or cells which express opioid receptorprotein by analyzing the mRNA, for example, using Northern blottechniques. Those tissues which are identified as containing mRNAencoding opioid receptor protein using the probes of the invention arethen suitable sources for preparation of cDNA libraries which mayfurther be probed using the cDNA described hereinbelow.

The DNA encoding the various vertebrate opioid receptor proteins,obtained in general as set forth above, according to the standardtechniques described hereinbelow, can be used to produce cells whichexpress the opioid receptor at their surface; such cells are typicallyeucaryotic cells, in particular, mammalian cells such as COS cells orCHO cells. Suitable expression systems in eucaryotic cells for suchproduction are described hereinbelow. The opioid receptor proteins mayalso be produced in procaryotes or in alternative eucaryotic expressionsystems for production of the protein per se. The DNA encoding theprotein may be ligated into expression vectors preceded by signalsequences to effect its secretion, or may be produced intracellularly,as well as at the cell surface, depending on the choice of expressionsystem and host. If desired, the opioid receptor protein thusrecombinantly produced may be purified using suitable means of proteinpurification, and, in particular, by affinity purification usingantibodies or fragments thereof immunospecific for the opioid receptorprotein.

Screening for Opioid Agonists and Antagonists Using Recombinant Cells

The ability of a candidate compound to act as an opicid agonist orantagonist may be assessed using the recombinant cells of the inventionin a variety of ways. To exhibit either agonist or antagonist activity,the candidate compound must bind to the opioid receptor. Thus, to assessthe ability of the candidate to bind, either a direct or indirectbinding assay may be used. For a direct binding assay, the candidatebinding compound is itself detectably labeled, such as with aradioisotope or fluorescent label, and binding to the recombinant cellsof the invention is assessed by comparing the acquisition of label bythe recombinant cells to the acquisition of label by correspondinguntransformed (control) cells.

More convenient, however, is the use of a competitive assay wherein thecandidate compound competes for binding to the recombinant cells of theinvention with a detectably labeled form of an opioid ligand known tobind to the receptor. Such ligands are themselves labeled usingradioisotopes or fluorescent moieties, for example. A particularlysuitable opioid known to bind to this receptor is diprenorphine. Atypical protocol for such an assay is as follows:

In general, about 10⁶ recombinant cells are incubated in suspension in1.0 ml of Kreb's Ringer Hepes Buffer (KRHB) at pH 7.4, 37° C. for 20 minwith ³H-diprenorphine. Nonspecific binding is determined by the additionof 400 nM diprenorphine in the binding mixtures. Various concentrationsof candidate compounds are added to the reaction mixtures. Theincubations are terminated by collecting the cells on Whatman GF-Bfilters, with removal of excess radioactivity by washing the filtersthree times with 5 ml of KRHB at 0° C. After incubating at 20° C.overnight in 5 ml of scintillation fluid, such as Liquiscint (NationalDiagnostics, Somerville, N.J.), the radioactivity on the filters isdetermined by liquid scintillation counting.

The K_(d) (dissociation constant) values for the candidate opiateligands can be determined from the IC₅₀ value (“inhibitoryconcentration₅₀” means the concentration of candidate ligand thatresults in a 50% decrease in binding of labeled diprenorphine).

The effects of sodium and GTP on the binding of ligands to therecombinantly expressed receptors can be used to distinguish agonistfrom antagonist activities. If the binding of a candidate compound issensitive to Na⁺ and GTP, it is more likely to be an agonist than anantagonist, since the functional coupling of opioid receptors to secondmessenger molecules such as adenylate cyclase requires the presence ofboth sodium and GTP (Blume et al., Proc. Natl. Acad. Sci. USA 73:26-35(1979)). Furthermore, sodium, GTP, and GTP analogues have been shown toeffect the binding of opioids and opioid agonists to opioid receptors(Blume, Life Sci 22:1843-52 (1978)). Since opioid antagonists do notexhibit binding that is sensitive to guanine nucleotides and sodium,this effect is used as a method for distinguishing agonists fromantagonists using binding assays.

In addition, agonist activity can directly be assessed by the functionalresult within the cell. For example, it is known that the binding ofopioid agonists inhibits cAMP formation, inhibits potassium channelactivation, inhibits calcium channel activation, and stimulates GTPase.Assessment of these activities in response to a candidate compound isdiagnostic of agonist activity. In addition, the ability of a compoundto interfere with the activating activity of a known agonist such asetorphine effectively classifies it as an antagonist.

In one typical assay, the measurement of cAMP levels in cells expressingopioid receptors is carried out by determining the amount of ³H-cAMPformed from intracellular ATP pools prelabeled with ³H-adenine (Law etal., supra). Thus, cAMP formation assays are carried out with 0.5×10⁶cells/0.5 ml of KRHB at pH 7.4, incubated at 37° C. for 20 minutes.After addition of the internal standard ³²P-cAMP, the radioactive cAMPis separated from other ³H-labeled nucleotides by known double-columnchromatographic methods. The opiate agonists' ability to inhibit cAMPaccumulation is then determined as described by Law et al. (supra).

The potency of a candidate opiate antagonist can be determined bymeasuring the ability of etorphine to inhibit cyclic AMP accumulation inthe presence and in the absence of known amounts of the candidateantagonist. The inhibition constant (K_(i)) of an antagonist can then becalculated from the equation for competitive inhibitors.

An interesting feature of screening assays using the prior art NG108-15cells is that the agonist adenylate cyclase inhibition functionapparently does not require binding of all receptors on these cells.

Thus, the K_(d) and K_(i) values for the opioid ligands differed whenusing these cells.

The foregoing assays, as described above, performed on the recombinantlytransformed cells of the present invention, provide a more direct andmore convenient screen for candidate compounds having agonist andantagonist opioid receptor activity than that previously available inthe art. Furthermore, such assays are more sensitive since cells can, inaccordance with the present invention, be engineered to express highlevels of the opioid receptor. Additionally, cells engineered inaccordance with the present invention will circumvent the concern thatNG108-15 cells, due to their tumor cell background, have a cellularenvironment that artifactually affects opioid receptor expression.

The mu opioid encoding DNA described herein also offer a means to followinheritance patterns. DNA sequence polymorphisms frequently occur in thenoncoding regions that surround genes. Polymorphisms are especiallyfrequent in repeat sequences such as CACACA which often show distinctpolymorphisms in the number of repeats that are present in differentindividuals. These polymorphisms offer a marker by which to follow theinheritance of the gene among family members. The inheritance of a gene(such as MORa) or its human counterpart can be followed by polymerasechain reaction (PCR) amplification of the region surrounding the CACACApolymorphism and analyzing the resulting products. This would be auseful diagnostic marker for the mu opioid receptor gene.

Methods to Preoare Opioid Receptor Protein or Portions Thereof

The present invention provides the amino acid sequence of a murineopioid receptor; similarly, the availability of the cDNA of theinvention places within possession of the art corresponding vertebrateopioid receptors whose amino acid sequence may also be determined bystandard methods. As the amino acid sequences of such opioid receptorsare known, or determinable, in addition to purification of such receptorprotein from native sources, recombinant production or synthetic peptidemethodology may also be employed for producing the receptor protein orpeptide.

The opioid receptor or portions thereof can thus also be prepared usingstandard solid phase (or solution phase) peptide synthesis methods, asis known in the art. In addition, the DNA encoding these peptides may besynthesized using commercially available oligonucleotide synthesisinstrumentation for production of the protein in the manner set forthabove. Production using solid phase peptide synthesis is, of course,required if amino acids not encoded by the gene are to be included.

The nomenclature used to describe the peptides and proteins of theinvention follows the conventional practice where the N-terminal aminogroup is assumed to be to the left and the carboxy group to the right ofeach amino acid residue in the peptide. In the formulas representingselected specific embodiments of the present invention, the amino- andcarboxy-terminal groups, although often not specifically shown, will beunderstood to be in the form they would assume at physiological pHvalues, unless otherwise specified. Thus, the N-terminal NH3⁺ andC-terminal COO⁻ at physiological pH are understood to be present thoughnot necessarily specified and shown, either in specific examples or ingeneric formulas. Free functional groups on the side chains of the aminoacid residues may also be modified by glycosylation, phosphorylation,cysteine binding, amidation, acylation or other substitution, which can,for example, alter the physiological, biochemical, or biologicalproperties of the compounds without affecting their activity within themeaning of the appended claims.

In the peptides shown, each gene-encoded residue, where appropriate, isrepresented by a single letter designation, corresponding to the trivialname of the amino acid, in accordance with the following conventionallist:

One-Letter Three-letter Amino Acid Symbol Symbol Alanine A Ala ArginineR Arg Asparagine N Asn Aspartic acid D Asp Cysteine C Cys Glutamine QGln Glutamic acid E Glu Glycine G Gly Histidine H His Isoleucine I IleLeucine L Leu Lysine K Lys Methionine M Met Phenylalanine F Phe ProlineP Pro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y TyrValine V Val

Nomenclature of Enkephalins

Enkephalins are either of two peptides having five residues with theN-terminal residue numbered 1:

tyr-gly-gly-phe-xxx (SEQ ID NO:18)  1   2   3   4   5

In “met enkephalin” the fifth residue is methionine:

tyr-gly-gly-phe-met (SEQ ID NO:1)

In “leu enkephalin” the 5th residue is leucine:

tyr-gly-gly-phe-leu (SEQ ID NO:2)

Enkephalin analogs can be made with (1) amino acid substitutions, (2)D-amino acid substitutions, and/or (3) additional amino acids. The siteat which the substitution is made is noted at the beginning of thecompound name. For example, “(D-ala², D-leu⁵) enkephalin” means thatD-ala is present at the second position and D-leu is present at thefifth position:

tyr-[D-ala]-gly-phe-[D-leu] (SEQ ID NO:5)

One letter abbreviations can also be used. Thus, “(D-ser²) leuenkephalin” could be abbreviated as “DSLE.” Additional residues arenoted as well. Thus, the addition of a threonine residue (to the sixthposition) of (D-ser²) leu enkephalin would be “(D-ser²) leu enkephalinthr” which could be abbreviated as “DSLET”:

tyr-[D-ser]-gly-phe-leu-thr (SEQ ID NO:3)

Antibodies

Antibodies immunoreactive with the opioid receptor protein or peptide ofthe present invention can be obtained by immunization of suitablemammalian subjects with peptides, containing as antigenic regions thoseportions of the receptor intended to be targeted by the antibodies.Certain protein sequences have been determined to have a high antigenicpotential. Such sequences are listed in antigenic indices, for example,MacVector software (I.B.I.) Thus, by determining the sequence of theopioid receptor protein and evaluating the sequence with an antigenicindex, probable antigenic sequences are located.

Antibodies are prepared by immunizing suitable mammalian hosts accordingto known immunization protocols using the peptide haptens alone, if theyare of sufficient length, or, if desired, or if required to enhanceimmunogenicity, conjugated to suitable carriers. Methods for preparingimmunogenic conjugates with carriers such as BSA, KLH, or other carrierproteins are well known in the art. In some circumstances, directconjugation using, for example, carbodiimide reagents may be effective;in other instances linking reagents such as those supplied by PierceChemical Co., Rockford, Ill., may be desirable to provide accessibilityto the hapten. The hapten peptides can be extended or interspersed withcysteine residues, for example, to facilitate linking to carrier.Administration of the immunogens is conducted generally by injectionover a suitable time period and with use of suitable adjuvants, as isgenerally understood in the art. During the immunization schedule,titers of antibodies are taken to determine adequacy of antibodyformation.

While the polyclonal antisera produced in this way may be satisfactoryfor some applications, for pharmaceutical compositions, use ofmonoclonal antibody (mAb) preparations is preferred. Immortalized celllines which secrete the desired mabs may be prepared using the standardmethod of Kohler and Milstein or modifications which effectimmortalization of lymphocytes or spleen cells, as is generally known.The immortalized cell lines secreting the desired mAbs are screened byimmunoassay in which the antigen is the peptide hapten or is the opioidreceptor itself displayed on a recombinant host cell. When theappropriate immortalized cell culture secreting the desired mAb isidentified, the cells can be cultured either in vitro or byintraperitoneal injection into animals wherein the mAbs are produced inthe ascites fluid.

The desired mAbs are then recovered from the culture supernatant or fromthe ascites fluid. In addition to intact antibodies, fragments of themAbs or of polyclonal antibodies which contain the antigen-bindingportion can be used as antagonists. Use of immunologically reactiveantigen binding fragments, such as the Fab, Fab′, of F(ab′)₂ fragments,is often preferable, especially in a therapeutic context, as thesefragments are generally less immunogenic than the whole immunoglobulinmolecule.

Standard Methods

The techniques for sequencing, cloning and expressing DNA sequencesencoding the amino acid sequences corresponding to a opioid receptor,e.g., polymerase chain reaction (PCR), synthesis of oligonucleotides,probing a cDNA library, transforming cells, constructing vectors,preparing antisense oligonucleotide sequences based on known sensenucleotide sequences, extracting messenger RNA, preparing cDNAlibraries, and the like are well-established in the art. Ordinarilyskilled artisans are familiar with the standard resource materials,specific conditions and procedures. The following paragraphs areprovided for convenience, it being understood that the invention islimited only by the appended claims.

RNA Preparation and Northern Blot

RNA preparation is as follows: The samples used for preparation of RNAare immediately frozen in liquid nitrogen and then stored until use at−80° C. The RNA is prepared by CsCl centrifugation (Ausubel et al.,supra) using a modified homogenization buffer (Chirgwin et al.,Biochemistry 18:5294-5299 (1979)). Poly(A⁺) RNA is selected by oligo(dT)chromatography (Aviv and Leder, Proc Natl Acad Sci USA 69:1408-1412(1972)). RNA samples are stored at −80° C.

Analysis of gene expression and tissue distribution can be accomplishedusing Northern blots with, e.g., radiolabeled probes. The mRNA issize-separated using gel electrophoresis and then typically istransferred to a nylon membrane or to nitrocellulose and hybridized withradiolabeled probe. Presence of the hybridized probe is detected usingautoradiography.

Cloning

The cDNA sequences encoding the opioid receptor protein are obtainedfrom a random-primed, size-selected cDNA library.

Alternatively, the cDNA sequences encoding opioid receptor protein areobtained from a cDNA library prepared from mRNA isolated from cellsexpressing the receptor protein in various organs such as the brain,according to procedures described in Sambrook, J. et al., MOLECULARCLONING: A LABORATORY MANUAL, 2nd Edition, Cold Spring Harbor Press,Cold Spring Harbor, N.Y., 1989.

The cDNA insert from the successful clone, excised with a restrictionenzyme such as EcoRI, is then used as a probe of the original cDNAlibrary or other libraries (low stringency) to obtain the additionalclones containing inserts encoding other regions of the protein thattogether or alone span the entire sequence of nucleotides coding for theprotein.

An additional procedure for obtaining cDNA sequences encoding the opioidreceptor protein is PCR. PCR is used to amplify sequences from a pooledcDNA library of reversed-transcribed RNA, using oligonucleotide primersbased on the transporter sequences already known.

Vector Construction

Construction of suitable vectors containing the desired coding andcontrol sequences employs ligation and restriction techniques which arewell understood in the art (Young et al., Nature 316:450-452 (1988)).Double-stranded cDNA encoding opioid receptor protein is synthesized andprepared for insertion into a plasmid vector CDM8. Alternatively,vectors such as Bluescript² or Lambda ZAP² (Stratagene, San Diego,Calif.) or a vector from Clontech (Palo Alto, Calif.) can be used inaccordance with standard procedures (Sambrook, J. et al., supra).

Site specific DNA cleavage is performed by treating with the suitablerestriction enzyme, such as EcoRI, or more than one enzyme, underconditions which are generally understood in the art, and theparticulars of which are specified by the manufacturer of thesecommercially available restriction enzymes. See, e.g., New EnglandBiolabs, Product Catalog. In general, about 1 μg of DNA is cleaved byone unit of enzyme in about 20 μl of buffer solution; in the examplesherein, typically, an excess of restriction enzyme is used to ensurecomplete digestion of the DNA substrate. Incubation times of about oneto two hours at about 37° C. are workable, although variations can betolerated. After each incubation, protein is removed by extraction withphenol/chloroform, and can be followed by other extraction and thenucleic acid recovered from aqueous fractions by precipitation withethanol.

In vector construction employing “vector fragments”, the vector fragmentis commonly treated with bacterial alkaline phosphatase (BAP) or calfintestinal alkaline phosphatase (CIP) in order to remove the 5′phosphate and prevent religation of the vector. Digestions are conductedat pH 8 in approximately 150 mM Tris, in the presence of Na⁺ and Mg⁺⁺using about 1 unit of BAP or CIP per μg of vector at 60° C. or 37° C.,respectively, for about one hour. In order to recover the nucleic acidfragments, the preparation is extracted with phenol/chloroform andethanol precipitated. Alternatively, religation can be prevented invectors which have been double digested by additional restriction enzymedigestion of the unwanted fragments.

Ligations are performed in 15-50 μl volumes under the following standardconditions and temperatures: 20 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 10 mMDTT, 33 μg/ml BSA, 10 mM to 50 mM NaCl, and either 40 μM ATP, 0.01-0.02(Weiss) units T4 DNA ligase at 0° C. (for “sticky end” ligation) or 1 mMATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14° C. (for “blunt end”ligation). Intermolecular “sticky end” ligations are usually performedat 33-100 μg/ml total DNA concentrations (5-100 nM total endconcentration). Intermolecular blunt end ligations (usually employing a10-30 fold molar excess of linkers) are performed at 1 μM total endsconcentration. Correct ligations for vector construction are confirmedaccording to the procedures of Young et al., Nature, 316:450-452 (1988).

cDNA Library Screening

cDNA libraries can be screened using reduced stringency conditions asdescribed by Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,Greene Publishing and Wiley-Interscience, New York (1990), or by usingmethods described in Sambrook et al. supra), or by using a colony orplaque hybridization procedure with a fragment of the DOR-1 cDNA codingfor opioid receptor protein.

Plaque hybridization is typically carried out as follows: Host bacteriasuch as LE 392 (Stratagene) are grown overnight at 37° in LB Broth(Sambrook et al., supra), gently pelleted and resuspended in one halfthe original volume of 10 mM MgSO₄, 10 mM CaCl². After titration, anamount of the phage library containing approximately 50,000 plaqueforming units (pfu) is added to 300 μl of the host bacteria, incubatedat 37° for 15 minutes and plated onto NZYCM agar with 10 ml NZYCM topagarose. A total of a million plaques distributed on twenty 15 cm platesare screened. For colony screening, transfected bacteria are plated ontoLB broth plates with the appropriate antibiotics. After the plaques orcolonies have grown to 1 mm, the plates are chilled at 4° C. for atleast two hours, and then overlaid with duplicate nitrocellulosefilters, followed by denaturation of the filters in 0.5 M NaOH/1.5 MNaCl for five minutes and neutralization in 0.5 M Tris, pH 7.4/1.5 MNaCl for five minutes. The filters are then dried in air, baked at 80°C. for two hours, washed in 5×SSC/0.5% SDS at 68° C. for several hours,and prehybridized in 0.5 M NaPO₄, pH 7.2/1% BSA/1 mM EDTA/7% SDS/100μg/ml denatured salmon sperm DNA for more than 4 hours. Using the DOR-1cDNA (described herein) labeled by random priming as a probe, highstringency hybridization is carried out in the same solution at 68° C.,and the temperature is reduced to 50-60° C. for lower stringencyhybridization. After hybridization for 16-24 hours, the filters arewashed first in 40 mM NaPO₄, pH 7.2/0.5% BSA/5% SDS/1 mM EDTA twice forone hour each, then in 40 mM NaPO₄, pH 7.2/1% BSA/1 mM EDTA for one houreach, both at the same temperature as the hybridization (Boulton et al.,Cell 65:663-675 (1991)). The filters are then exposed to film with anenhancing screen at −70° C. for one day to one week.

Positive signals are then aligned to the plates, and the correspondingpositive phage is purified in subsequent rounds of screening, using thesame conditions as in the primary screen. Purified phage clones are thenused to prepare phage DNA for subcloning into a plasmid vector forsequence analysis. Tissue distribution of DNA corresponding to thevarious independent clones is analyzed using Northern blots and in situhybridization using standard methods. Function of the DNA is testedusing expression in a heterologous eucaryotic expression system such asCOS cells.

Expression of Opioid Receptor Protein

The nucleotide sequence encoding opioid receptor protein can beexpressed in a variety of systems. The cDNA can be excised by suitablerestriction enzymes and ligated into procaryotic or eucaryoticexpression vectors for such expression.

For example, as set forth below, the cDNA encoding the protein isexpressed in COS cells. To effect functional expression, the plasmidexpression vector CDM8 (Aruffo and Seed, Proc Natl Acad Sci USA84:8573-8577 (1987), provided by Drs. Aruffo and Seed (HarvardUniversity, Boston, Mass.) was used. Alternatively, other suitableexpression vectors such as retroviral vectors can be used.

Procaryotic and prefereably eucaryotic systems can be used to expressthe opioid receptor. Eucaryotic microbes, such as yeast, can be used ashosts for mass production of the opioid receptor protein. Laboratorystrains of Saccharomyces cerevisiae, Baker's yeast, are used most,although a number of other strains are commonly available. Vectorsemploying, for example, the 2 μ origin of replication (Broach, Meth.Enz. 101:307 (1983)), or other yeast compatible origins of replications(e.g., Stinchcomb et al., Nature 282:39 (1979)); Tschempe et al., Gene10:157 (1980); and Clarke et al., Meth. Enz. 101:300 (1983)) can beused. Control sequences for yeast vectors include promoters for thesynthesis of glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149(1968); Holland et al., Biochemistry 17:4900 (1978)). Additionalpromoters known in the art include the promoter for 3-phosphoglyceratekinase (Hitzeman et al., J. Biol. Chem. 255:2073 (1980)), and those forother glycolytic enzymes. Other promoters, which have the additionaladvantage of transcription controlled by growth conditions are thepromoter regions for alcohol dehydrogenase 2, isocytochrome C, acidphosphatase, degradative enzymes associated with nitrogen metabolism,and enzymes responsible for maltose and galactose utilization. It isalso believed terminator sequences are desirable at the 3′ end of thecoding sequences. Such terminators are found in the 3′ untranslatedregion following the coding sequences in yeast-derived genes.

Alternatively, genes encoding opioid receptor protein are expressed ineucaryotic host cell cultures derived from multicellular organisms.(See, e.g., Tissues Cultures, Academic Press, Cruz and Patterson, eds,(1973)). These systems have the additional advantage of the ability tosplice out introns, and thus can be used directly to express genomicfragments. Useful host cell lines include amphibian cocytes such asXenopus oocytes, COS cells, VERO and HeLa cells, Chinese hamster ovary(CHO) cells, and insect cells such as SF9 cells. Expression vectors forsuch cells ordinarily include promoters and control sequences compatiblewith mammalian cells such as, for example, the commonly used early andlate promoters from baculovirus, vaccinia virus, Simian Virus 40 (SV40)(Fiers et al., Nature 273:113 (1973)), or other viral promoters such asthose derived from polyoma, Adenovirus 2, bovine papilloma virus, oravian sarcoma viruses. The controllable promoter, hMTII (Karin et al.,Nature 299:797-802 (1982)) may also be used. General aspects ofmammalian cell host system transformations have been described by Axel,U.S. Pat. No. 4,399,216. It now appears, that “enhancer” regions areimportant in optimizing expression; these are, generally, sequencesfound upstream or downstream of the promoter region in non-coding DNAregions. Origins of replication can be obtained, if needed, from viralsources. However, integration into the chromosome is a common mechanismfor DNA replication in eucaryotes.

If procaryotic systems are used, an intronless coding sequence should beused, along with suitable control sequences. The cDNA of opioid receptorprotein can be excised using suitable restriction enzymes and ligatedinto procaryotic vectors along with suitable control sequences for suchexpression.

Procaryotes most frequently are represented by various strains of E.coli; however, other microbial species and strains may also be used.Commonly used procaryotic control sequences which are defined herein toinclude promoters for transcription initiation, optionally with anoperator, along with ribosome binding site sequences, including suchcommonly used promoters as the β-lactamase (penicillinase) and lactose(lac) promoter systems (Chang et al., Nature 198:1056 (1977)) and thetryptophan (trp) promoter system (Goeddel et al., Nucl Acids Res 8:4057(1980)) and the λ derived P_(L) promoter and N-gene ribosome bindingsite (Shimatake et al., Nature 292:128 (1981)).

Depending on the host cell used, transformation is carried out usingstandard techniques appropriate to such cells. The treatment employingcalcium chloride, as described by Cohen, Proc Natl Acad Sci USA (1972)69:2110 (1972) or by Sambrook et al. (supra), can be used forprocaryotes or other cells which contain substantial cell wall barriers.For mammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology 54:546 (1978),optionally as modified by Wigler et al., Cell 16:777-785 (1979), or byChen and Okayama, supra, can be used. Transformations into yeast can becarried out according to the method of Van Solingen et al., J. Bact.130:946 (1977), or of Hsiao et al., Proc Natl Acad Sci USA 76:3829(1979).

Other representative transfection methods include viral transfection,DEAE-dextran mediated transfection techniques, lysozyme fusion orerythrocyte fusion, scraping, direct uptake, osmotic or sucrose shock,direct microinjection, indirect microinjection such as viaerythrocyte-mediated techniques, and/or by subjecting host cells toelectric currents. The above list of transfection techniques is notconsidered to be exhaustive, as other procedures for introducing geneticinformation into cells will no doubt be developed.

Modulation of Expression by Antisense Sequences

Alternatively, antisense sequences may be inserted into cells expressingopioid receptors as a means to modulate functional expression of thereceptors encoded by sense oligonucleotides. The antisense sequences areprepared from known sense sequences (either DNA or RNA), by standardmethods known in the art. Antisense sequences specific for the opioidreceptor gene or RNA transcript can be used to bind to or inactivate theoligonucleotides encoding the opioid receptor.

Terminology

As used herein, the singular forms “a”, “an” and “the” include pluralreference unless the context clearly dictates otherwise. Thus, forexample, reference to “a receptor” includes mixtures of such receptors,reference to “an opioid” includes a plurality of and/or mixtures of suchopioids and reference to “the host cell” includes a plurality of suchcells of the same or similar type and so forth.

Unless defined otherwise all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The following examples areintended to illustrate but not to limit the invention. Temperatures arein ° C. and pressures at near atmospheric unless otherwise specified.

Preparation of Mono ¹²⁵I-DADLE

DADLE (Peninsula Laboratories Inc.) was iodinated using the iodogenmethod (Maidment et al., in: MICRODIALYSIS IN THE NEUROSCIENCES, T.Robinson and J. Justice, eds., pp. 275-303 (Elsevier, 1991)). Both mono-and di-iodinated forms are produced. It has been reported thatdi-iodo-DADLE does not bind opiate receptors, due to the di-iodinationof the tyrosine residue (Miller, R. J., et al., Life Sci. 22:379-88(1978)). Accordingly, mono-iodinated DADLE is preferred. Mono-¹²⁵I-DADLEis also preferred because it has extremely high specific activitycompared to DADLE labeled with other isotopes. Thus, exposure times onthe order of days, rather than weeks or months can be used.

By employing a molar ratio of sodium iodide to peptide of approximately1:100 when carrying out iodination, the yield of the preferredmono-iodinated DADLE was increased. Additionally, to further enhance theyield of the mono-iodinated form, iodinated DADLE (containing both mono-and di-iodinated forms) was purified by reverse-phase HPLC (Maidment etal., supra). Employing this procedure a single major radiolabeled peakof the mono-iodinated DADLE separated from di-iodinated andnon-iodinated forms.

DADLE monolabeled with ¹²⁵I is crucial to successful screening.Radiolabeled ¹²⁵I-DADLE differs from DADLE in several importantparameters: size, hydrophobicity, and binding affinity (slightly lower).The purification of mono-iodinated from di-iodinated and non-iodinatedDADLE by the HPLC step yields a ligand with very high specific activity(approximately 2000 Ci/mmol). The specific activity of themono-iodinated form is approximately 100 times greater than thatobtained by using the unseparated mixture of mono-, di-, andnon-iodinated DADLE. Monolabeled ¹²⁵I-DADLE must be used within a fewdays of its preparation.

Example 1

Preparation of DOR-1

The NG108-15 cell line (available from Dr. Christopher Evans, UCLA)comprises a homogeneous and enriched source of delta opioid receptors.Utilizing mRNA isolated from NG108-15, a random-primed, size-selectedcDNA library was constructed in plasmid vector CDM8. The cDNA librarywas amplified in bacteria. The cDNA library was transfected into COS-7cells by electroporation. Transiently transfected COS lawns werescreened and selected with highly purified mono-¹²⁵I-2dAla, 5dLeuenkephalin (¹²⁵I-DADLE). Positive clones were identified by filmautoradiography, and plasmids from these cells were recovered andamplified in bacteria. Thereafter, the plasmids were re-transfected intoCOS cells. Following three cycles of such plasmid enrichment, individualclones were transfected and a pure clone was identified that bound¹²⁵I-DADLE.

A. Construction of the cDNA Library

RNA was prepared from NG108-15 cells by homogenization in 6 Mguanidinium isothiocyanate, followed by centrifugation through cesiumchloride (J. M. Chirgwin, et al., Biochemistry 18:5294 (1979)). Poly-A⁺RNA was isolated by chromatography over oligo-dT-cellulose (H. Aviv andP. Leder, Proc Natl Acad Sci USA 69:1408 (1972)). Using this RNA as atemplate, random hexamers were used to prime cDNA synthesis by avianmyeloblastosis virus reverse transcriptase (Life Sciences Inc.). Secondstrand synthesis was accomplished with RNase-H and E. coli DNApolymerase (U. Gubler and B. J. Hoffman, Gene 24: 263 (1983)). The endsof the cDNAs were rendered blunt with T4 DNA polymerase and BstXIlinkers were added. cDNA longer than 1.5 kb was selected byelectrophoresis through 5% acrylamide followed by electro-elution. The1.5 kb cDNA was ligated to the CDM8 vector (A. Aruffo and B. Seed,supra, and then transformed into MC-1061 bacteria by electroporation (W.J. Dower et al., Nucl. Acids Res. 16:6127 (1988)). Accordingly, sixpools of plasmid DNA were prepared from the original cDNA library ofapproximately 2×10⁶ recombinants.

B. Plasmid Transfection by Electroporation and Expression in COS cells

COS cells were grown at high density and were harvested in trypsin, thenresuspended at 2×10⁷/ml in 1.2× RPMI containing 20% fetal calf serum.These cells were then incubated for ten minutes at 4° C. with 20 μgrecombinant plasmid DNA from the cDNA library described above, and thenelectroporated at 960 μF and 230 V in a 0.4 cm gap cuvette (BioRad). Thecells were then incubated an additional ten minutes at 4° C., and thenplated into Dulbecco's Modified Eagle's Medium (DMEM) plus 10 fetal calfserum (FCS).

C. Screening of Transfected COS Cells

The transfected COS cells as obtained above were grown for three days,then screened using radiolabeled mono ¹²⁵I-DADLE. Transfected COS lawnswere washed with PBS, then incubated at room temperature with 10-20 nM¹²⁵I-DADLE in KHRB containing 1% BSA. After 1 hour, the plates werewashed rapidly several times with ice cold PBS then dried on ice withstrong flow of forced cold air. Plates were exposed on Dupont Cronexfilm in cassettes at room temperature. Positive clones were identifiedby careful alignment of the film with the petri dish via low powermicroscopy.

DNA was removed from positive cells by solubilization in 0.1% SDS in TEcontaining 1 μg/μl tRNA delivered from a syringe attached to a capillarytube on a micromanipulator. Plasmids were purified from the extractedcells using the Hirt lysis procedure (Hirt, B., J. Mol. viol. 26:365-369(1967)), and electroporated into MC-1061 bacteria. The plasmids werepurified then retransfected into COS cells. Following three suchenriching cycles, individual plasmid clones were transfected into COScells yielding a single clone, named the DOR-1 clone.

Example 2

Characterization of DOR-1

The DOR-1 clone initially was characterized by screening cell membranefractions, from cells expressing DOR-1, with the labelled DADLE it wasfound that binding of ¹²⁵I DADLE was displaced by nanomolarconcentrations of opiate alkaloids diprenorphine, morphine, etorphine,and by DADLE, DSLET and DPDPE. Dextrorphan (10 μM) did not displace the¹²⁵I DADLE, whereas its opioid-active enantiomer levorphanol diddisplace the radiolabeled DADLE. Additionally, the mu receptor-selectiveligand DAGO (5 μM) did not displace the counts.

The DOR-1 clone was further characterized pharmacologically by assessingbinding of ³H-diprenorphine to intact cells expressing the DOR-1 clone(FIG. 1), and by assessing displacement of ³H-diprenorphine frommembrane fractions of such cells (FIGS. 2 and 3).

Binding assays were conducted on intact cells in KRHB, 1% BSA; or onmembranes in 25 mM HEPES, 5 mM MgCl₂ pH 7.7. Cells were harvested withPBS containing 1 mM EDTA, washed 2× with PBS then resuspended in KHRB.Membranes prepared from the cells (Law P. Y. E et al., Mol. Pharm.23:26-35 (1983)) were used directly in the binding assay. Binding assayswere conducted in 96 well polypropylene cluster plates (Costar), at 4°C. in a total volume of 100 μl with an appropriate amount ofradiolabeled ligand. Following 1 hour of incubation, plates wereharvested on a Tomtec harvester and “B” type filtermats were counted ina Betaplate (Pharmacia) scintillation counter using Meltilex B/HS(Pharmacia) melt-on scintillator sheets.

Intact cells expressing DOR-1 were analyzed with the high affinityopiate antagonist ³H-diprenorphine. Specific binding was defined by thecounts displaced by 400 nM diprenorphine. FIG. 1 shows a saturationcurve for ³H-diprenorphine for NG108-15 cells, and COS-7 cellstransfected with the delta opioid receptor clone. Untransfected COScells, or COS cells transfected with plasmid having no insert showed nospecific binding. Thus, the opioid binding of COS-DOR-1 cells wassimilar to that of NG108-15 cells.

Membranes prepared by standard methods from transfected COS-7 cells wereemployed for a more extensive pharmacological characterization of thereceptor encoded by the DOR-1 clone. The affinities for the followingalkaloid opiates in competition for ³H-diprenorphine are illustrated inFIG. 2: unlabeled diprenorphine, a high affinity antagonist for deltareceptors; etorphine, a high affinity agonist for delta, mu and kappareceptors; levorphanol, a low affinity agonist for delta receptors;morphine, a low affinity agonist for delta receptors and a high affinityagonist for mu receptors; and dextrorphan, a non-opiate activeenantiomer of levorphanol which should not bind delta receptors.

As shown in FIG. 2, the displacement of ³H-diprenorphine, in decreasingorder of affinity, was observed with diprenorphine, etorphine,levorphanol and morphine. As expected, ³H-diprenorphine was notdisplaced by dextrorphan.

The affinities of the following opioid peptides in competition for³H-diprenorphine are set forth in FIG. 3: DADLE, a high affinity agonistfor mu and delta receptors; DSLET and DPDPE, both high affinity agonistsof delta (but not mu) receptors; DAGO, a selective agonist for mureceptors; and Dynorphin 1-17, a high affinity agonist for kappareceptors and moderate to low affinity agonist for delta receptors. Asshown in FIG. 3, the displacement of ³H-diprenorphine, in decreasingorder of affinity, was observed for DSLET, DPDPE and DADLE, andDynorphin 1-17. Only weak displacement by DAGO was observed.

Example 3

Northern Blot Analysis of RNA

For Northern analysis, the mRNA from NG108-15 cells, and from cellsdissected from regions of rat brain was separated by electrophoresisthrough 2.2 M formaldehyde/1.5% agarose, blotted to nylon and hybridizedin aqueous solution at high stringency. The filters were prehybridizedin 0.5 M NaPO₄, pH 7.2; 1% BSA; 1 mM EDTA; 7% SDS; and 100 μg/mldenatured salmon sperm DNA for at least four hours at 68° C. (Boulton etal., supra). The filters were then hybridized overnight under these sameconditions with a ≧5×10⁶ cpm/ml purified cDNA insert labelled by randompriming (A. P. Feinberg and B. Vogelstein, Anal. Biochem. 132:6 (1983)).The filters were twice washed in 40 mM NaPO₄, pH 7.2; 0.5% BSA; 5% SDS;and 1 mM EDTA for one hour, and then washed twice in 40 mM NaPO₄, pH7.2; 1% SDS; and 1 mM EDTA for one hour each, all at 68° C. Thereafterautoradiography was performed with DuPont Cromex Lightening Plus at −70°C.

The results of the Northern analysis of the mRNA showed the presence ofmultiple bands hybridizing to the probe at approximately 8.7, 6.8, 4.4,2.75 and 2.2 kilobases (Kb) (FIG. 4). Also, the Northern analysisindicates that the pattern of mRNA may vary between brain regions. Atpresent, it is unclear whether these mRNAs encode different proteinsequences, and if so, whether these messages represent different typesor sub-types of opioid receptors.

Example 4

Southern Blot Analysis of DNA

The radiolabeled DOR-1 cDNA probe was hybridized to genomic Southernblots by standard methods (Sambrook et al., supra). Accordingly, theradiolabeled DOR-1 cDNA probe was hybridized under high stringencyconditions to a blot of NG108-15, mouse, rat and human DNA cut withrestriction endonuclease BamHI (FIG. 7). Single bands were observed inthe clones containing the NG108-15, mouse, and rat DNA. The sizes of thebands hybridizing to the cDNA probe were estimated to be 5.2 kb(NG108-15), 5.2 kb (mouse), and 5.7 kb (rat). These results indicate theclose homology of the mouse and rat genes, and also demonstrate that theDOR-1 clone is from the murine parent of the NG108-15 cell line.

In a blot containing EcoRI-cut genomic DNA from many different species,hybridization of the DOR-1 cDNA under conditions of moderate stringencyshowed two bands in each lane of mouse, rat, human, rabbit, and severalother mammalian species. This demonstrates a close relationship betweenopioid receptor genes in all of these species. Further, these resultsshow that the genes or cDNAs from each of these species may readily becloned using hybridization under moderate stringency.

Example 5

Determination of the cDNA Sequence

Isolated cDNA represented by the DOR clone was analyzed by subcloningthe insert from the cDNA clone into a plasmid such as pBluescript™(Stratagene, San Diego, Calif.) and using the dideoxy method (Sanger etal., Proc Natl Acad Sci USA 74:5463-5467 (1977)). The sequence of thecDNA was determined from single-stranded EDNA and specifically designedinternal primers, using both Sequenase and ΔTaq cycle sequencing kits(USB). These kits, widely used in the art, utilize the dideoxy chaintermination method. The DNA sequence and predicted protein sequence wasthen compared to sequences in established databanks such as GenBank.

Sequencing the cDNA insert in the DOR-1 clone, revealed an open readingframe of 370 amino acids (FIG. 5). Comparisons with known sequences inGenBank showed highest homology between DOR-1 and the G-protein-coupledsomatostatin receptor (57% amino acid identity), and slightly lowerhomology with the receptors binding angiotensin, the two chemotacticfactors IL-8 and N-formyl peptide. FIG. 6 shows the homology to thehuman somatostatin 1 receptor. The close homology of the presentreceptor clone with the somatostatin receptor is especially noteworthysince somatostatin ligands are reported to bind to opioid receptors, andto have molecular mechanisms similar to those in delta receptors.

Other features of the DOR-1 clone amino acid sequence deduced from thecDNA sequence include three consensus glycosylation sites at residues 18and 33 (predicted to be in the extracellular N-terminal domain), and atresidue 310 (close to the C-terminus and predicted to be intracellular).Phosphokinase C consensus sites are present within predictedintracellular domains, at residues 242, 255, 344, and 352. Sevenputative membrane-spanning regions were identified based onhydrophobicity profiles, as well as homology with Rhodopsin and otherG-protein coupled receptors which have been analyzed with respect tomembrane-spanning regions using MacVector (I.B.I.) analysis. The DOR-1clone isolated in accordance with the principles of the presentinvention produces a delta receptor with a predicted molecular weight of40,558 daltons prior to post-translational modifications such asN-glycosylation.

Example 6

Isolation of Opioid Receptor Genomic Clones

Isolation of genomic clones was carried out according to techniquesknown in the art. To isolate opiate receptor genomic clones, 300,000human genomic clones in γgem 11 (Promega) and a similar number of mousegenomic clones in lambda Fix (Stratagene) were plated on host strainLe392 and probed with the 1.1 kb DOR-1 Pst/Xba I fragment, whichcontains primarily the coding region. The conditions for hybridizationwere of fairly low stringency: 50% formamide/6×SSC, overnight at 37° C.The washes were performed also at low stringency: 2×SSC, 0.1 SDS at roomtemperature.

One mouse clone and three human genomic clones were isolated andpurified by sequential rounds of hybridization and plaque purification.DNA preparation and restriction analysis showed that the three humanclones had very different EcoRI digestion patterns. The 1.1 kb opiatereceptor probe hybridized to a different single EcoRI band in Southernblot analysis for each clone. These results indicated preliminarily thatthree different genes were represented by the human genomic clones whichwere designated H3, H14 and H20 (see FIGS. 8a, 8 b, 8 c and 8 d). Eachof these clones was deposited on Aug. 13, 1993 at the American TypeCulture Collection, Rockville, Md., under conditions of the BudapestTreaty. All restrictions on access to these deposits will be irrevocablyremoved at the time a patent issues in the United States on the basis ofthis application. The ATCC deposit numbers are 75549 for H20; 75550 forH14 and 75551 for H3.

The H3, H14 and H20 clones were digested into smaller fragments by EcoRIand TaqI and then shotgun cloned into the appropriate site of Bluescriptfor sequencing. The partial nucleotide sequence for H3 is shown in FIG.8a; the partial nucleotide sequence of H14 is shown in FIG. 8b; thepartial nucleotide sequence of H20 is shown in FIG. 8c.

The three genomic clones were mapped by in situ hybridization on humanmetaphase chromosomes by Dr. Glenn Evans of the Salk Institute. H3 mapsto chromosome 1P; H14 maps near the centromere of chromosome 8, and H20maps to chromosome 6. Comparison of sequence data obtained as describedabove with the published sequences for the murine counterpartsreferenced hereinabove, and with the DOR-2 clone described hereinbelow,confirmed that: (a) H3 encodes the human delta opioid receptor; (b) H14encodes the human kappa opioid receptor and (c) H20 encodes the human mureceptor. In addition, H20 appears to contain a CACACA marker (FIG. 8d)which provides a means to track the inheritance of this gene.

The genomic clones were digested into smaller fragments by EcoRI andTaqI, then shotgun cloned into the appropriate site of Bluescript forsequencing.

Example 7

Isolation of Opioid Receptor Clones From Additional Organisms

In order to isolate the opioid receptor from mammalian brain cells, forexample human brain cells, a random-primed human brainstem cDNA libraryin λ Zap (Stratagene) was screened using the murine cDNA encoding theDOR-1 described herein. Positive plaques were purified and rescreened.Individual positive clones are sequenced and characterized as above.

Example 8

Determination of Probable Antigenic Sequences

By evaluating the amino acid sequence of the opioid receptor encoded byDOR-1 with the MacVector (I.B.I.) antigenic index, and the antigenicindex in accordance to Jameson, B. and H. Wolf, Comput. Applic. inBiosci. 4:181-186 (1988), the following underlined sequences of thedelta opioid receptor were determined to have a high antigenicpotential:

NH ₂ MELVPSARAELOSSPLVNLSDAFPSAFPSAGANASGSPGARSASSLALAIAITALYSAVCAVGLLGNVLVMFGIVRYTKLKTATNIYIFNLALADALATSTLPFQSAKYLMETWPFGELLCKAVLSIDYYNMFTSIFTLTMMSVDRYIAVCHPVKALDFRTPAKAKLINICIWVLASGVGVPIMVMAVTQPRDGAVVCMLQFPSPSWYWDTVTKICVFLFAFVVPILIITVCYGLMLLRLRSVRLLSGSKEKDRSLRRITRMVLVVVGAFVVCWAPIHIFVIVWTLVDINRRDPLVVAALHLCIALGYANSSLNPVLYAFLDENFKRCFRQLCRTPCGROEPGSLRRPROATTRERVT ACTPSDGPGGGAAA-COOH (SEQID NO:2).

The N-terminal sequence is extracellular, the other four sequences arepredicted to be intracellular.

Example 9

Recovery of the Murine Clone DOR-2 (mMOR-1)

A cDNA library prepared from mouse brain in λgt10 was probed using thelow-stringency conditions of Example 6 using DOR-1 as a probe. One clonewas recovered, inserted into Bluescript and sequenced. Northern andSouthern blots indicated divergence from DOR-1. This clone, designatedDOR-2, represented a new gene. DOR-2 hybridized to a different patternof neurons than did DOR-1 and showed greater labeling of the striatum.Expression of DOR-1 by insertion into the vector pCDNA and transfectioninto mammalian cells produced cells which bind morphine, indicative of amu-receptor. The cells also bind the nonselective opiate antagonistdiprenorphine. The identity of DOR-2 (mMOR-1) as that of a mu receptorwas confirmed by the displacement of ³H-DPN by nanomolar concentrationsof the mu-selective ligands morphiceptin, DAMGO and morphine. The deltaselective ligands DPDPE and deltorphan did not displace the binding andnaloxone had the expected high affinity. The partial sequence designatedH20, described in Example 6, was substantially similar to DOR-2. Thepartial sequence of DOR-2 is shown in FIG. 9.

FIG. 10 shows a comparison of the amino acid sequences of murine deltareceptin with the rat mu and kappa receptors. There are extensiveregions of homology.

18 5 amino acids amino acid single linear not provided 1 Tyr Gly Gly PheMet 1 5 5 amino acids amino acid single linear not provided 2 Tyr GlyGly Phe Leu 1 5 6 amino acids amino acid single linear not providedModified-site /note= “D form of amino acid” 3 Tyr Ser Gly Phe Leu Thr 15 5 amino acids amino acid single linear not provided Modified-sitegroup(2, 5) /product= “OTHER” /note= “D-penicillamine” 4 Tyr Xaa Gly PheXaa 1 5 5 amino acids amino acid single linear not providedModified-site /note= “D form of amino acid” Modified-site /note= “D formof amino acid” 5 Tyr Ala Gly Phe Leu 1 5 5 amino acids amino acid singlelinear not provided Modified-site /note= “D form of amino acid”Modified-site /product= “MePhe” /note= “N-Methylphenylalanine”Modified-site /product= “Gly-ol” /note= “Carboxy end of glycine has beenreplaced with an alcohol substituent” 6 Tyr Ala Gly Xaa Xaa 1 5 1829base pairs nucleic acid single linear not provided CDS 29..1144 7GCACGGTGGA GACGGACACG GCGGCGCC ATG GAG CTG GTG CCC TCT GCC CGT 52 MetGlu Leu Val Pro Ser Ala Arg 1 5 GCG GAG CTG CAG TCC TCG CCC CTC GTC AACCTC TCG GAC GCC TTT CCC 100 Ala Glu Leu Gln Ser Ser Pro Leu Val Asn LeuSer Asp Ala Phe Pro 10 15 20 AGC GCC TTC CCC AGC GCG GGC GCC AAT GCG TCGGGG TCG CCG GGA GCC 148 Ser Ala Phe Pro Ser Ala Gly Ala Asn Ala Ser GlySer Pro Gly Ala 25 30 35 40 CGT AGT GCC TCG TCC CTC GCC CTA GCC ATC GCCATC ACC GCG CTC TAC 196 Arg Ser Ala Ser Ser Leu Ala Leu Ala Ile Ala IleThr Ala Leu Tyr 45 50 55 TCG GCT GTG TGC GCA GTG GGG CTT CTG GGC AAC TGTCTC GTC ATG TTT 244 Ser Ala Val Cys Ala Val Gly Leu Leu Gly Asn Cys LeuVal Met Phe 60 65 70 GGC ATC GTC CGG TAC ACC AAA TTG AAG ACC GCC ACC AACATC TAC ATC 292 Gly Ile Val Arg Tyr Thr Lys Leu Lys Thr Ala Thr Asn IleTyr Ile 75 80 85 TTC AAT CTG GCT TTG GCT GAT GCG CTG GCC ACC AGC ACG CTGCCC TTC 340 Phe Asn Leu Ala Leu Ala Asp Ala Leu Ala Thr Ser Thr Leu ProPhe 90 95 100 CAG AGC GCC AAG TAC TTG ATG GAA ACG TGG CCG TTT GGC GAGCTG CTG 388 Gln Ser Ala Lys Tyr Leu Met Glu Thr Trp Pro Phe Gly Glu LeuLeu 105 110 115 120 TGC AAG GCT GTG CTC TCC ATT GAC TAC TAC AAC ATG TTCACT AGC ATC 436 Cys Lys Ala Val Leu Ser Ile Asp Tyr Tyr Asn Met Phe ThrSer Ile 125 130 135 TTC ACC CTC ACC ATG ATG AGC GTG GAC CGC TAC ATT GCTGTC TGC CAT 484 Phe Thr Leu Thr Met Met Ser Val Asp Arg Tyr Ile Ala ValCys His 140 145 150 CCT GTC AAA GCC CTG GAC TTC CGG ACA CCA GCC AAG GCCAAG CTG ATC 532 Pro Val Lys Ala Leu Asp Phe Arg Thr Pro Ala Lys Ala LysLeu Ile 155 160 165 AAT ATA TGC ATC TGG GTC TTG GCT TCA GGT GTC GGG GTCCCC ATC ATG 580 Asn Ile Cys Ile Trp Val Leu Ala Ser Gly Val Gly Val ProIle Met 170 175 180 GTC ATG GCA GTG ACC CAA CCC CGG GAT GGT GCA GTG GTATGC ATG CTC 628 Val Met Ala Val Thr Gln Pro Arg Asp Gly Ala Val Val CysMet Leu 185 190 195 200 CAG TTC CCC AGT CCC AGC TGG TAC TGG GAC ACT GTGACC AAG ATC TGC 676 Gln Phe Pro Ser Pro Ser Trp Tyr Trp Asp Thr Val ThrLys Ile Cys 205 210 215 GTG TTC CTC TTT GCC TTC GTG GTG CCG ATC CTC ATCATC ACG GTG TGC 724 Val Phe Leu Phe Ala Phe Val Val Pro Ile Leu Ile IleThr Val Cys 220 225 230 TAT GGC CTC ATG CTA CTG CGC CTG CGC AGC GTG CGTCTG CTG TCC GGT 772 Tyr Gly Leu Met Leu Leu Arg Leu Arg Ser Val Arg LeuLeu Ser Gly 235 240 245 TCC AAG GAG AAG GAC CGC AGC CTG CGG CGC ATC ACGCGC ATG GTG CTG 820 Ser Lys Glu Lys Asp Arg Ser Leu Arg Arg Ile Thr ArgMet Val Leu 250 255 260 GTG GTG GTG GGC GCC TTC GTG GTG TGC TGG GCG CCCATC CAC ATC TTC 868 Val Val Val Gly Ala Phe Val Val Cys Trp Ala Pro IleHis Ile Phe 265 270 275 280 GTC ATC GTC TGG ACG CTG GTG GAC ATC AAT CGGCGC GAC CCA CTT GTG 916 Val Ile Val Trp Thr Leu Val Asp Ile Asn Arg ArgAsp Pro Leu Val 285 290 295 GTG GCC GCA CTG CAC CTG TGC ATT GCG CTG GGCTAC GCC AAC AGC AGC 964 Val Ala Ala Leu His Leu Cys Ile Ala Leu Gly TyrAla Asn Ser Ser 300 305 310 CTC AAC CCG GTT CTC TAC GCC TTC CTG GAC GAGAAC TTC AAG CGC TGC 1012 Leu Asn Pro Val Leu Tyr Ala Phe Leu Asp Glu AsnPhe Lys Arg Cys 315 320 325 TTC CGC CAG CTC TGT CGC ACG CCC TGC GGC CGCCAA GAA CCC GGC AGT 1060 Phe Arg Gln Leu Cys Arg Thr Pro Cys Gly Arg GlnGlu Pro Gly Ser 330 335 340 CTC CGT CGT CCC CGC CAG GCC ACC ACG CGT GAGCGT GTC ACT GCC TGC 1108 Leu Arg Arg Pro Arg Gln Ala Thr Thr Arg Glu ArgVal Thr Ala Cys 345 350 355 360 ACC CCC TCC GAC GGC CCG GGC GGT GGC GCTGCC GCC TGACCTACCC 1154 Thr Pro Ser Asp Gly Pro Gly Gly Gly Ala Ala Ala365 370 GACCTTCCCC TTAAACGCCC CTCCCAAGTG AAGTGATCAG AGGCCACACCGAGCTCCCTG 1214 GGAGGCTGTG GCCACCACCA GGACAGCTAG AATTGGGCCT GCACAGAGGGGAGGCCTCCT 1274 GTGGGGACGG GCCTGAGGGA TCAAAGGCTC CAGGTTGGAA CGGTGGGGGTGAGGAAGCAG 1334 AGCTGGTGAT TCCTAAACTG TATCCATTAG TAAGGCCTCT CAATGGGACAGAGCCTCCGC 1394 CTTGAGATAA CATCGGGTTC TGGCCTTTTT GAACACCCAG CTCCAGTCCAAGACCCAAGG 1454 ATTCCAGCTC CAGAACCAGG AGGGGCAGTG ATGGGGTCGA TGATTTGGTTTGGCTGAGAG 1514 TCCCAGCATT TGTGTTATGG GGAGGATCTC TCATCTTAGA GAAGAAAGGGGACAGGGCAT 1574 TCAGGCAAGG CAGCTTGGGG TTTGGTCAGG AGATAAGCGC CCCCCTTCCCTTGGGGGGAG 1634 GATAAGTGGG GGATGGTCAC GTTGGAGAAG AGTCAAAGTT CTCACCACCTTTCTAACTAC 1694 TCAGCTAAAC TCGTTGAGGC TAGGGCCAAC GTGACTTCTC TGTAGAGAGGTACAAGCCGG 1754 GCCTGATGGG GCAGGCCTGT GTAATCCCAG TCATAGTGGA GGCTGAGGCTGGAAAATTAA 1814 GGACCAACAG CCCGG 1829 372 amino acids amino acid linearprotein not provided 8 Met Glu Leu Val Pro Ser Ala Arg Ala Glu Leu GlnSer Ser Pro Leu 1 5 10 15 Val Asn Leu Ser Asp Ala Phe Pro Ser Ala PhePro Ser Ala Gly Ala 20 25 30 Asn Ala Ser Gly Ser Pro Gly Ala Arg Ser AlaSer Ser Leu Ala Leu 35 40 45 Ala Ile Ala Ile Thr Ala Leu Tyr Ser Ala ValCys Ala Val Gly Leu 50 55 60 Leu Gly Asn Cys Leu Val Met Phe Gly Ile ValArg Tyr Thr Lys Leu 65 70 75 80 Lys Thr Ala Thr Asn Ile Tyr Ile Phe AsnLeu Ala Leu Ala Asp Ala 85 90 95 Leu Ala Thr Ser Thr Leu Pro Phe Gln SerAla Lys Tyr Leu Met Glu 100 105 110 Thr Trp Pro Phe Gly Glu Leu Leu CysLys Ala Val Leu Ser Ile Asp 115 120 125 Tyr Tyr Asn Met Phe Thr Ser IlePhe Thr Leu Thr Met Met Ser Val 130 135 140 Asp Arg Tyr Ile Ala Val CysHis Pro Val Lys Ala Leu Asp Phe Arg 145 150 155 160 Thr Pro Ala Lys AlaLys Leu Ile Asn Ile Cys Ile Trp Val Leu Ala 165 170 175 Ser Gly Val GlyVal Pro Ile Met Val Met Ala Val Thr Gln Pro Arg 180 185 190 Asp Gly AlaVal Val Cys Met Leu Gln Phe Pro Ser Pro Ser Trp Tyr 195 200 205 Trp AspThr Val Thr Lys Ile Cys Val Phe Leu Phe Ala Phe Val Val 210 215 220 ProIle Leu Ile Ile Thr Val Cys Tyr Gly Leu Met Leu Leu Arg Leu 225 230 235240 Arg Ser Val Arg Leu Leu Ser Gly Ser Lys Glu Lys Asp Arg Ser Leu 245250 255 Arg Arg Ile Thr Arg Met Val Leu Val Val Val Gly Ala Phe Val Val260 265 270 Cys Trp Ala Pro Ile His Ile Phe Val Ile Val Trp Thr Leu ValAsp 275 280 285 Ile Asn Arg Arg Asp Pro Leu Val Val Ala Ala Leu His LeuCys Ile 290 295 300 Ala Leu Gly Tyr Ala Asn Ser Ser Leu Asn Pro Val LeuTyr Ala Phe 305 310 315 320 Leu Asp Glu Asn Phe Lys Arg Cys Phe Arg GlnLeu Cys Arg Thr Pro 325 330 335 Cys Gly Arg Gln Glu Pro Gly Ser Leu ArgArg Pro Arg Gln Ala Thr 340 345 350 Thr Arg Glu Arg Val Thr Ala Cys ThrPro Ser Asp Gly Pro Gly Gly 355 360 365 Gly Ala Ala Ala 370 369 aminoacids amino acid single linear not provided 9 Met Glu Leu Thr Ser GluGln Phe Asn Gly Ser Gln Val Trp Ile Pro 1 5 10 15 Ser Pro Phe Asp LeuAsn Gly Ser Leu Gly Pro Ser Asn Gly Ser Asn 20 25 30 Gln Thr Glu Pro TyrTyr Asp Met Thr Ser Asn Ala Val Leu Thr Phe 35 40 45 Ile Tyr Phe Val ValCys Val Val Gly Leu Cys Gly Asn Thr Leu Val 50 55 60 Ile Tyr Val Ile LeuArg Tyr Ala Lys Met Lys Thr Ile Thr Asn Ile 65 70 75 80 Tyr Ile Leu AsnLeu Ala Ile Ala Asp Glu Leu Phe Met Leu Gly Leu 85 90 95 Pro Phe Leu AlaMet Gln Val Ala Leu Val His Trp Pro Phe Gly Lys 100 105 110 Ala Ile CysArg Val Val Met Thr Val Asp Gly Ile Asn Gln Phe Thr 115 120 125 Ser IlePhe Cys Leu Thr Val Met Ser Ile Asp Arg Tyr Leu Ala Val 130 135 140 ValHis Pro Ile Lys Ser Ala Lys Trp Arg Arg Pro Arg Thr Ala Lys 145 150 155160 Met Ile Asn Val Ala Val Trp Gly Val Ser Leu Leu Val Ile Leu Pro 165170 175 Ile Met Ile Tyr Ala Gly Leu Arg Ser Asn Gln Trp Gly Arg Ser Ser180 185 190 Cys Thr Ile Asn Trp Pro Gly Glu Ser Gly Ala Trp Tyr Thr GlyPhe 195 200 205 Ile Ile Tyr Ala Phe Ile Leu Gly Phe Leu Val Pro Leu ThrIle Ile 210 215 220 Cys Leu Cys Tyr Leu Phe Ile Ile Ile Lys Val Lys SerSer Gly Ile 225 230 235 240 Arg Val Gly Ser Ser Lys Arg Lys Lys Ser GluLys Lys Val Thr Arg 245 250 255 Met Val Ser Ile Val Val Ala Val Phe IlePhe Cys Trp Leu Pro Phe 260 265 270 Tyr Ile Phe Asn Val Ser Ser Val SerVal Ala Ile Ser Pro Thr Pro 275 280 285 Ala Leu Lys Gly Met Phe Asp PheVal Val Ile Leu Thr Tyr Ala Asn 290 295 300 Ser Cys Ala Asn Pro Ile LeuTyr Ala Phe Leu Ser Asp Asn Phe Lys 305 310 315 320 Lys Ser Phe Gln AsnVal Leu Cys Leu Val Lys Val Ser Gly Ala Glu 325 330 335 Asp Gly Glu ArgSer Asp Ser Lys Gln Asp Lys Ser Arg Leu Asn Glu 340 345 350 Thr Thr GluThr Gln Arg Thr Leu Leu Asn Gly Asp Leu Gln Thr Ser 355 360 365 Ile 130base pairs nucleic acid single linear not provided 10 GGGCAGTGGTGTGCATGCTC CAGTTCCCCA GCCCCAGCTG GTACTGGGAC ACGGTGACCA 60 AGATCTGCGTGTTCCTCTTC GCCTTCGTGG TGCCCATCCT CATCATCACC GTGTGCTATG 120 GCCTCATGCT130 130 base pairs nucleic acid single linear not provided 11 GGTGCAGTGGTATGCATGCT CCAGTTCCCC AGTCCCAGCT GGTACTGGGA CACTGTGACC 60 AAGATCTGCGTGTTCCTCTT TGCCTTCGTG GTGCCGATCC TCATCATCAC GGTGTGCTAT 120 GGCCTCATGC130 2447 base pairs nucleic acid single linear not provided 12CCTGGCCTTT TGGGGATGTG CTGTGCAAGA TAGTAATTTC CATTGATTAC TACAACATGT 60TCACCAGCAT CTTCACCTTG ACCATGATGA GCGTGGACCG CTACATTGCC GTGTGCCACC 120CCGTGAAGGC TTTGGACTTC CGCACACCCT TGAAGGCAAA GATCATCAAT ATCTGCATCT 180GGCTGCTGTC GTCATCTGTT GGCATCTCTG CAATAGTCCT TGGAGGCACC AAAGTCAGGG 240AAGGTAAGAG CAGTCATTTC ATTCTGTTCA TAAAAATGTA GCTTCAAATT ACATAGACTT 300TTAATTTGAG CGTGAGTAGG CCACATATTT GTGGAAATCG ATGCCAAAAG ACGACGGAAA 360TGTAGTGCCT AAATCCATGG AAGATGAGAA GTAGAACAAT TTTTTGTCCC TTTCCACCTC 420TAAACACAGA ATGCAATAAT GACATTGCCA GAAGAGAGAT GCCCGACCTG TCTCCCATTC 480TGGCAATGTT TAGTAGAAAG TGGAGGGGTG AGGATGAGGT AAGAACCACA GGCATGTAGA 540TTTTAAAGTA CAACCTGGCA AGTCCAGACA CACCTTCTCA CTCCTTTTTT TCTCTTTAAC 600AAGGGATATA AATTATTGGT GACATATGCT GGTTGTTTCC TCTTTTATTC CTAAAGGATA 660ACCTCCAAAT CACTATTTTA ACAGCTTTGG CGTAGGATCT CAAAATCAAG TTAACGGATG 720GTAGTTACAG ATGAGTCAGA ACCACTTGAT TTGGACATAT CAGGTTTTCC CTTGCAAACC 780AGCCAACTGA TTTTTTTTTT TTTTTTTTTT GAGAGAGAGT CTTGCTCTGT TGCCAGGCTA 840GAGTGCAGTG GCGCGATATC GGCTCACTGC AACCTCTGCC TCCCGGGTTC AACCTCAGCC 900TCTCGAGTAG CTGGGACTAC TGGCACACAC CACCATGCCC AGCTAATTTT TGTATTTTTA 960GTAGAGACAG GGTTTCACCG TGTTGGCCAG GGTGGTCTCA ATCTCTTGAC CTCGTGATCT 1020GCCCGCCTCG NCTCCCCAAA GTGCTGGGAT TACAGGCGTG CNCTGCNCCC GNCCCCTGTT 1080GATGTTTTTC CTGTATTTCT AGGACAGTAG TTCTCACTCT GGGCTGCACA TTGGAATCAC 1140CTGGGTACTT TAGAAAACAC TGCTGCCTGC ATCCCACCCC TTAAGGGTCT GGTGTAATTG 1200ACCTGGGGTA CAGCCTGGGT GTCAAGATTT TTGAGCTCTC TCCAGGTGAC TCTGACCTGC 1260AGCCAAGGTG AGAGGTACTG TTCTAGGAGT TTTGCTTTAC TAGCAAAATA TAAAGCTATA 1320GAAAGCATCT TTTGTTCCTC ATAGAAATTA ATGATGGGGA GGTGAGCAGA ATAGTCACTC 1380TGGGCCTACT CATGCTGTTT AATGCTCCAG CAGGTATATA GGTTCTCCAG TTACTAGGGG 1440GTTCATAATA CCTGTGAGAG CAGATAACTG AGTGTATATA GTGAGGATTT CCAGGTCATA 1500GTGAAAGGGC AAGGCACTAA AATCATAGCT TGTCTTGCAT ATACTGTTTG TTTGTTTTTA 1560GACTTACATG TTAGGTTTCA GTTTACGTTT TAGGTTCACA GCAAAACTGA CCAGAAAGCA 1620CAGAGAGGCA CTTCNATTTA CCTCCATTTA CCCCACACAG GCACATCCTC CCCTACAGAG 1680TGGTCCATTT ATTACAGCTG CTGAACCCAC ACTGACACGC TGTTATCACT CAGAGCCTGG 1740CAGTTTACAG AGGCTCACTC TCCGNTATGT GTCCTGTGNT TTGAACAAAT GTATAATGAC 1800TTTATTCATT GTTTTTTAAT GAAGCTGATC TTTTCCCTCT GAAACTACAA AATGAATTTC 1860TAGCATAGCC ATAGCAGGTG TCAAGCTATA CTACTAGGTA AATTTTAAGA AATGCCCAAC 1920TTTATCATAT TTGCATTTCA AAATATGATT AATCACACAT AGGATTTTGT TTCTTCATGC 1980CTACAGCAAA TAGAAATAAA GTGCAAGAAA CTTTTCTGAG GCAAAGCTTT CACTTTGTGA 2040ACGTAAAATG TTGACTCTAA TATTTTCCAT ACTGTAGTAT ATGTGTGTGT ATTATGTGAG 2100GATTCATAGT CTGCTCTTAC TTTTTTATAG TAGCTAAGAA TTATTATAAT CGCTATAAGC 2160AGAAACAATT ATTCTTAACA AAATGAATAC ACACAAGAAA AGCTTTAGTT TAGCTATTAG 2220AACTAACTCT ATAATTATGA TAACCATGAG ATGCTGGAAC AGGAGCCAGC AGAAGCCACA 2280GCCCTCTGAT ATTAATATAT AAAGAAACCA AAATCTGCTT GTTAAACTGA GGCAGTTGTA 2340TGGATACTTC AACCTGAAAA TGCCCCCTTC TTCCTGAAAC AGAACATTTA ATAAAAATGG 2400CATGCTTGGA CAGGAATTTC TTTTTTAAAA AATGCTTAGT TTTTATG 2447 830 base pairsnucleic acid single linear not provided 13 TTCCTTTATC TCCTAGATACACCAAGATGA AGACTGCCAC CAACATCTAC ATTTTCAACC 60 TTGCTCTGCA GATGCCTTAGCCACCAGTAC CCTGCCCTTC CAGAGTGTGA ATTACCTAAT 120 GGGAACATGG CCATTTGGAACCATCCTTTG CAAGATAGTG ATCTCCATAG ATTACTATAA 180 CATGTTCACC AGCATATTCACCCTCTGCAC CATGAGTGTT GATCGATACA TTGCAGTCTG 240 CCACCCTGTC AAGGCCTTAGATTTCCGTAC TCCCCNNNNN NNNNNNNNNN NNNNNNNNNN 300 NNNNNNNNNN NNNNNNNNNNNNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 360 NNNNNNNNGT TCCATAGATTGTACACTAAC ATTCTCTCAT CCAACCTGGT ACTGGGAAAA 420 CCTGCTGAAG ATCTGTGTTTTCATCTTCGC CTTCATTATG CCAGTGCTCA TCATTACCGT 480 GTGCTATGGA CTGATGATCTTGCGCCTCAA GAGTGTCCGC ATGCTCTCTG GCTCCAAAGA 540 AAAGGACAGG AATCTTCGAAGGATCACCAG GATGGTGCTG GTGGTGGTGG CTGTGTTCAT 600 CGTCTGCTGG ACTCCCATTCACATTTACGT CATCATTAAA GCCTTGGTTA CAATCCCAGA 660 AACTACGTTC CAGACTGTTTCTTGGCACTT CTGCATTGCT CTAGGTTACA CAAACAGCTG 720 CCTCAACCCA GTCCTTTATGCATTTCTGGA TGAAAACTTC CACGATGCTT CAGAGAGTTC 780 TGTATCCCAA CCTCTTCCAACATTGAGCAA CAAAACTCCA CTCGAATTCC 830 332 base pairs nucleic acid singlelinear not provided 14 GGGTACCGGG CCCCCCCTCG AGGTCGACGG TATCGATAAGCTTGATATCG AATTCTTACT 60 GAATTAGGTA TCTTTCTTCA CACTACTTGG TAAAAAAAATGAAAAGGCAG AAAAATTAGC 120 CCCAAAAGAG ATGAAACTCT TCCGTCCATC ACCATTGACTCTATTGTGAA CTTATGAAAA 180 AGGTAGTTGA GCAATATGAA GGCCATGATG TGGAATTAAACACACACACA CACACACACA 240 CACACACACA CACATGCTGG ATTCTAAATG TGTCCTTCCTCCTCTCACTC TCTTGATTCA 300 AGTTTATTTC TGAACTGAGA CACGATCACC AC 332 1981base pairs nucleic acid single linear not provided 15 CGGATCCTTAGCATCCCCAA AGCGCCTCCG TGTACTTCTA AGGTGGGAGG GGGATACAAG 60 CAGAGGAGAATATCGGACGC TCAGACGTTC CATTCTGCCT GCCGCTCTTC TCTGGTTCCA 120 CTAGGGCTTGTCCTTGTAAG AAACTGACGG AGCCTAGGGC AGCTGTGAGA GGAAGAGGCT 180 GGGGCGCCTGGAACCCGAAC ACTCTTGAGT GCTCTCAGTT ACAGNCTACC GAGTCCGCAG 240 GAAGCATTCAGAACCATGGA CAGCAGCGCC GGCCCAGGGA ACATCAGCGA CTGCTCTGAC 300 CCCTTAGCTCCTGCAAGTTG CTCCCCAGCA CCTGGCTCCT GGCTCAACTT GTCCCACGTT 360 GATGGAAACCAGTCCGACCC ATGCGGTCCT AACCCGACGG GCCTTGGCGG GAACGACAGC 420 CTGTGCCCTCAGACCGGCAG CCCTTCCATG GTCACAGCCA TCACCATCAT GGCCCTCTAT 480 TCTATCGTGTGTGTAGTGGG CCTCTTTGGA AACTTCCTGG TCATGTATGT GATTGTAAGA 540 TATACCAAAATGAAGACTGC CACCAACATC TACATTTTCA ACCTTGCTCT GGCAGATGCC 600 TTAGCCACTAGCACGCTGCC CTTTCAGAGT GTTAACTACC TGATGGGAAC GTGGCCCTTT 660 GGAAACATCCTCTGCAAGAT CGTGATCTCA ATAGACTACT ACAACATGTT CACCAGTATC 720 TTCACCCTCTGCACCATGAG TGTAGACCGC TACATTGCCG TCTGCCACCC GGTCAAGGCC 780 CTGGATTTCCGTACCCCCCG AAATGCCAAA ATTGTCAATG TCTGCAACTG GATCCTCTCT 840 TCTGCCATTGGTCTGCCCGT AATGTTCATG GCAACCACAA AATACAGGCA GGGGTCCATA 900 GATTGCACCCTCACGTTCTC TCATCCCACA TGGTACTGGG AGAACCTGCT CAAAATCTGT 960 GTCTTCATCTTCGCCTTCAT CATGCCGGGC CTCATCATCA CTGTGTGTTA TGGACTGATG 1020 ATCTTACAGCTCAAGAGTGT CCGCATGCTG TCGGGCTCCA AAGAAAAGGA CAGGAACCTG 1080 CGCAGGATCACCCGGATGGT GCTGGTGGTC GTGGCTGTAT TTATTGTCTG CTGGACCCCC 1140 ATCCACATCTATGTCATCAT CAAAGCACTG ATCACGATTC CAGAAACCAC TTTCCAGACT 1200 GTTTCCTGGCACTTCTGCAT TGCCTTGGGT TACACAAACA GCTGCCTGAA CCCAGTTCTT 1260 TATGCGTTCCTGGATGAAAA CTTCAAACGA TGTTTTAGAG AGTTCTGCAT CCCAACTTCC 1320 TCCACAATCGAACAGCAAAA CTCTGCTCGA ATCCGTCAAA ACACTAGGGA ACACCCCTCC 1380 ACGGCTAATACAGTGGATCG AACTAACCAC CAGCTAGAAA ATCTGGAAGC AGAAACTGCT 1440 CCATTGCCCTAACTGGGTCC CACGCCATCC AGACCCTCGC TAAACTTAGA GGCTGCCATC 1500 TACTTGGAATCAGGTTGCTG TCAGGGTTTG TGGGAGGCTC TGGTTTCCTG GAAAAGCATC 1560 TGATCCTGCATCATTCAAAG TCATTCCTCT CTGGCTATTC ACGCTACACG TCAGAGACAC 1620 TCAGACTGTGTCAAGCACTC AGAAGGAAGA GACTGCAGGC CACTACTGAA TCCAGCTCAT 1680 GTACAGAAACATCCAATGGA CCACAATACT CTGTGGTATG TGATTTGTGA TCAACATAGA 1740 AGGTGACCCTTCCCTATGTG GAATTTTTAA TTTCAAGGAA ATACTTATGA TCTCATCAAG 1800 GGAAAAATAGATGTCACTTG TTAAATTCAC TGTAGTGATG CATAAAGGAA AAGCTACCTC 1860 TGACCTCTAGCCCAGTCACC CTCTATGGAA AGTTCCATAG GGAATATGTG AGGGAAAATG 1920 TTGCTTCCAAATTAAATTTT CACCTTTATG TTATAGTCTA GTTAAGACAT CAGGGGCATC 1980 T 1981 398amino acids amino acid single linear not provided 16 Met Asp Ser Ser ThrGly Pro Gly Asn Thr Ser Asp Cys Ser Asp Pro 1 5 10 15 Leu Ala Gln AlaSer Cys Ser Pro Ala Pro Gly Ser Trp Leu Asn Leu 20 25 30 Ser His Val AspGly Asn Gln Ser Asp Pro Cys Gly Leu Asn Arg Thr 35 40 45 Gly Leu Gly GlyAsn Asp Ser Leu Cys Pro Gln Thr Gly Ser Pro Ser 50 55 60 Met Val Thr AlaIle Thr Ile Met Ala Leu Tyr Ser Ile Val Cys Val 65 70 75 80 Val Gly LeuPhe Gly Asn Phe Leu Val Met Tyr Val Ile Val Arg Tyr 85 90 95 Thr Lys MetLys Thr Ala Thr Asn Ile Tyr Ile Phe Asn Leu Ala Leu 100 105 110 Ala AspAla Leu Ala Thr Ser Thr Leu Pro Phe Gln Ser Val Asn Tyr 115 120 125 LeuMet Gly Thr Trp Pro Phe Gly Thr Ile Leu Cys Lys Ile Val Ile 130 135 140Ser Ile Asp Tyr Tyr Asn Met Phe Thr Ser Ile Phe Thr Leu Cys Thr 145 150155 160 Met Ser Val Asp Arg Tyr Ile Ala Val Cys His Pro Val Lys Ala Leu165 170 175 Asp Phe Arg Thr Pro Arg Asn Ala Lys Ile Val Asn Val Cys AsnTrp 180 185 190 Ile Leu Ser Ser Ala Ile Gly Leu Pro Val Met Phe Met AlaThr Thr 195 200 205 Lys Tyr Arg Gln Gly Ser Ile Asp Cys Thr Leu Thr PheSer His Pro 210 215 220 Thr Trp Tyr Trp Glu Asn Leu Leu Lys Ile Cys ValPhe Ile Phe Ala 225 230 235 240 Phe Ile Met Pro Ile Leu Ile Ile Thr ValCys Tyr Gly Leu Met Ile 245 250 255 Leu Arg Leu Lys Ser Val Arg Met LeuSer Gly Ser Lys Glu Lys Asp 260 265 270 Arg Asn Leu Arg Arg Ile Thr ArgMet Val Leu Val Val Val Ala Val 275 280 285 Phe Ile Val Cys Trp Thr ProIle His Ile Tyr Val Ile Ile Lys Ala 290 295 300 Leu Ile Thr Ile Pro GluThr Thr Phe Gln Thr Val Ser Trp His Phe 305 310 315 320 Cys Ile Ala LeuGly Tyr Thr Asn Ser Cys Leu Asn Pro Val Leu Tyr 325 330 335 Ala Phe LeuAsp Glu Asn Phe Lys Arg Cys Phe Arg Glu Phe Cys Ile 340 345 350 Pro ThrSer Ser Thr Ile Glu Gln Gln Asn Ser Thr Arg Val Arg Gln 355 360 365 AsnThr Arg Glu His Pro Ser Thr Ala Asn Thr Val Asp Arg Thr Asn 370 375 380His Gln Leu Glu Asn Leu Glu Ala Glu Thr Ala Pro Leu Pro 385 390 395 376amino acids amino acid single linear not provided 17 Met Glu Ser Pro IleGln Ile Phe Arg Gly Asp Pro Gly Pro Thr Cys 1 5 10 15 Ser Pro Ser AlaCys Leu Leu Pro Asn Ser Ser Ser Trp Phe Pro Asn 20 25 30 Trp Ala Glu SerAsp Ser Asn Gly Ser Val Gly Ser Glu Asp Gln Gln 35 40 45 Leu Glu Ser AlaHis Ile Ser Pro Ala Ile Pro Val Ile Ile Thr Ala 50 55 60 Val Tyr Ser ValVal Phe Val Val Gly Leu Val Gly Asn Ser Leu Val 65 70 75 80 Met Phe ValIle Ile Arg Tyr Thr Lys Met Lys Thr Ala Thr Asn Ile 85 90 95 Tyr Ile PheAsn Leu Ala Leu Ala Asp Ala Leu Val Thr Thr Thr Met 100 105 110 Pro PheGln Ser Ala Val Tyr Leu Met Asn Ser Trp Pro Phe Gly Asp 115 120 125 ValLeu Cys Lys Ile Val Ile Ser Ile Asp Tyr Tyr Asn Met Phe Thr 130 135 140Ser Ile Phe Thr Leu Thr Met Met Ser Val Asp Arg Tyr Ile Ala Val 145 150155 160 Cys His Pro Val Lys Ala Leu Asp Phe Arg Thr Pro Leu Lys Ala Lys165 170 175 Ile Ile Asn Ile Cys Ile Trp Leu Leu Ala Ser Ser Val Gly IleSer 180 185 190 Ala Ile Val Leu Gly Gly Thr Lys Val Arg Glu Asp Val IleGlu Cys 195 200 205 Ser Leu Gln Phe Pro Asp Asp Glu Trp Trp Asp Leu PheMet Lys Ile 210 215 220 Cys Val Phe Val Phe Ala Phe Val Ile Pro Val LeuIle Ile Ile Val 225 230 235 240 Cys Tyr Thr Leu Met Ile Leu Arg Leu LysSer Val Arg Leu Leu Ser 245 250 255 Gly Ser Arg Glu Lys Asp Arg Asn LeuArg Arg Ile Thr Lys Leu Val 260 265 270 Leu Val Val Val Ala Val Phe IleIle Cys Trp Thr Pro Ile His Ile 275 280 285 Phe Ile Leu Val Glu Ala LeuGly Ser Thr Ser His Ser Thr Ala Ala 290 295 300 Leu Ser Ser Tyr Tyr PheCys Ile Ala Leu Gly Tyr Thr Asn Ser Ser 305 310 315 320 Leu Asn Pro ValLeu Tyr Ala Phe Leu Asp Glu Asn Phe Lys Arg Cys 325 330 335 Phe Arg AspPhe Cys Phe Pro Ile Lys Met Arg Met Glu Arg Gln Ser 340 345 350 Thr AsnArg Val Arg Asn Thr Val Gln Asp Pro Ala Ser Met Arg Asp 355 360 365 ValGly Gly Met Asn Lys Pro Val 370 375 5 amino acids amino acid singlelinear not provided Modified-site /note= “Any amino acid” 18 Tyr Gly GlyPhe Xaa 1 5

What is claimed is:
 1. A nucleic acid probe that hybridizes to thecomplement of SEQ ID NO:12 under high stringency conditions, whereinsaid high stringency conditions are defined as washing twice in 40 mMNaPO₄, pH 7.2; 0.5% BSA; 5% SDS; and 1 mM EDTA for one hour at 68° C.followed by washing twice in 40 mM NaPO₄, pH 7.2; 1% SDS; and 1 mM EDTAfor one hour at 68° C. and specifically identifies a mammalian kappaopioid receptor clone in a cDNA library.
 2. A nucleic acid probe thathybridizes under high stringency conditions to the complement of SEQ IDNO:13 or 15, wherein said high stringency conditions are defined aswashing twice in 40 mM NaPO₄, pH 7.2; 0.5% BSA; 5% SDS; and 1 mM EDTAfor one hour at 68° C. followed by washing twice in 40 mM NaPO₄, pH 7.2;1% SDS; and 1 mM EDTA for one hour at 68° C. and specifically identifiesa mammalian mu opioid receptor clone in a cDNA library.
 3. An isolatedand purified or recombinant DNA molecule containing a nucleotidesequence encoding a mammalian kappa opioid receptor which nucleotidesequence hybridizes under conditions of high stringency, wherein saidhigh stringency conditions are defined as washing twice in 40 mM NaPO₄,pH 7.2; 0.5% BSA; 5% SDS; and 1 mM EDTA for one hour at 68° C. followedby washing twice in 40 mM NaPO₄, pH 7.2; 1% SDS; and 1 mM EDTA for onehour at 68° C., to a probe consisting of the nucleotide sequence SEQ IDNO: 12 or to its complement.
 4. A DNA molecule comprising an expressionsystem which, when transformed into a host, produces a mnammalian kappaopioid receptor in the host, which expression system comprises anucleotide sequence encoding said opioid receptor operably linked toheterologous control sequences operable to effect expression of saidnucleotide sequence in said host, wherein said opioid receptor isdefined as encoded by a nucleotide sequence which hybridizes underconditions of high stringency to the nucleotide sequence SEQ ID NO: 12or to its complement, wherein said high stringency conditions aredefined as washing twice in 40 mM NaPO₄, pH 7.2; 0.5% BSA; 5% SDS; and 1mM EDTA for one hour at 68° C. followed by washing twice in 40 mMNaPOa₄, pH 7.2; 1% SDS; and 1 mM EDTA for one hour at 68° C. 5.Recombinant host cells modified to contain the expression system ofclaim
 4. 6. A method to produce recombinant cells that display mammaliankappa opioid receptors at their surface, which method comprisesculturing the cells of claim 5 under conditions that effect expressionof the encoding nucleotide sequence to produce said receptors at theirsurface.
 7. An isolated ad purified or recombinant DNA moleculecontaining a nucleotide sequence encoding a mammalian mu opioid receptorwhich nucleotide sequence hybridizes under conditions of highstringency, wherein said high stringency conditions are define aswashing twice in 40 mM NaPO₄, pH 7.2; 0.5% BSA; 5% SDS; and 1 mM EDTAfor one hour at 68° C. followed by washing twice in 40 mM NaPO⁴, pH 7.2;1% SDS; and 1 mM EDTA for one hour at 68° C., to a probe consisting ofthe nucleotide sequence SEQ ID NO: 13 or 15 or to its complement.
 8. ADNA molecule comprising an expression system which, when transformredinto a host, produces a mammalian mu opioid receptor in the host, whichexpression system comprises a nucleotide sequence encoding said opioidreceptor operably linked to heterologous control sequences operable toeffect expression of said nucleotide sequence in said host, wherein saidopioid receptor is defined as encoded by a nucleotide sequence whichhybridizes under conditions of high stringency to the nucleotidesequence SEQ ID NO: 13 or 15 or to its complement, wherein said highstringency conditions are defined as washing twice in 40 mM NaPO₄, pH7.2; 0.5% BSA; 5% SDS; and 1 mM EDTA for one hour at 68° C. followed bywashing twice in 40 mM NaPO₄, pH 7.2; 1% SDS; and 1 mM EDTA for one hourat 68° C.
 9. Recombinant host cells modified to contain the expressionsystem of claim
 8. 10. A method to produce recornbinant cells thatdisplay mammalian mu opioid receptors at their surface, which methodcomprises culturing the cells of claim 9 under conditions that effectexpression of the encoding nucleotide sequence to produce said receptorsat their surface.